Aldolases, Nucleic Acids Encoding Them and Methods for Making and Using Them

ABSTRACT

This invention relates to polypeptides having aldolase activity, including pyruvate activity such as, without limitation, HMG and/or KHG aldolase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. In some embodiments, the invention is directed to polypeptides having aldolase activity, including pyruvate activity such as, without limitation, HMG and/or KHG aldolase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides in accordance with the invention can be used in a variety of pharmaceutical, agricultural and industrial contexts. In some embodiments, the invention provides polypeptides and biosynthetic pathways that are useful in the production of R-2-hydroxy 2-(indol-3ylmethyl)-4-keto glutaric acid (R-MP) and certain stereoisomers of monatin, such as R,R and S,R monatin, and salts thereof, as well as certain stereoisomers of monatin derivatives, such as the R,R and S,R configurations, and salts thereof.

FIELD IN ACCORDANCE WITH THE INVENTION

This invention relates to molecular and cellular biology andbiochemistry. More specifically, the invention relates to polypeptideshaving aldolase activity, polynucleotides encoding these polypeptides,and methods of making and using these polynucleotides and polypeptides.

BACKGROUND OF THE INVENTION

Monatin is a high-intensity sweetener having the chemical formula:

Monatin includes two chiral centers leading to four potentialstereoisomeric configurations. The R,R configuration (the “R,Rstereoisomer” or “R,R monatin”); the S,S configuration (the “S,Sstereoisomer” or “S,S monatin”); the R,S configuration (the “R,Sstereoisomer” or “R,S monatin”); and the S,R configuration (the “S,Rstereoisomer” or “S,R monatin”). As used herein, unless statedotherwise, the term “monatin” is used to refer to compositions includingall four stereoisomers of monatin, compositions including anycombination of monatin stereoisomers, (such as a composition includingonly the R,R and S,S, stereoisomers of monatin), as well as a singleisomeric form.

For purposes of this disclosure, the monatin carbon backbone will benumbered as illustrated above, with the carbon directly covalentlyattached to the alcohol group being identified as the 2-position carbonand the carbon directly covalently attached to the amino group beingidentified as the 4-position carbon. Consequently, references herein toR,R monatin, S,S monatin, R,S monatin, and S,R monatin mean: 2R,4Rmonatin, 2S,4S monatin, 2R,4S monatin, and 2S,4R monatin, respectively,unless otherwise indicated.

It should be noted that in the literature, the monatin carbon backbonehas also been numbered using an alternative convention, with the carbonattached to the alcohol group being the 4-position carbon, and thecarbon attached to the amino group being the 2-position carbon.Accordingly, for example, references to 2S,4R monatin in this disclosurewould be the same as references to 2R,4S monatin in literature using thealternative numbering convention.

Furthermore, because of various naming conventions, monatin is known bya number of alternative chemical names, including:2-hydroxy-2-(indol-3-ylmethyl)-4-aminoglutaric acid;4-amino-2-hydroxy-2-(1H-indol-3-ylmethyl)-pentanedioic acid;4-hydroxy-4-(3-indolylmethyl)glutamic acid; and,3-(1-amino-1,3-dicarboxy-3-hydroxy-but-4-yl)indole.

Certain isomeric forms of monatin can be found in the bark of roots ofthe Schlerochiton ilicifolius plant located in the Transvaal Region ofSouth Africa. U.S. patent application Ser. Nos. 10/422,366 (“the '366application”) and 10/979,821 (“the '821 application”), which are herebyincorporated by reference, disclose, inter alia, polypeptides, pathways,and microorganisms for in vitro and in vivo production of monatin.

SUMMARY

The invention provides polypeptides having aldolase activity(hereinafter “aldolases”), including pyruvate aldolase activity such as,without limitation, HMG and KHG aldolase activity, polynucleotidesencoding the polypeptides, and methods for making and using thepolypeptides and polynucleotides. In some embodiments, the inventionalso provides compositions (such as pharmaceutical compositions, fueland fuel additive compostions, foods and food additives, beverage andbeverage additives, feeds and feed additives, drugs and drug additives,dietary supplements) comprising the polypeptides or polynucleotides inaccordance with the invention. These compositions can be formulated in avariety of forms, such as tablets, gels, pills, implants, liquids,sprays, films, micelles, powders, food, feed pellets or as any type ofencapsulated form.

In some embodiments, the aldolases and/or compositions thereof may beuseful in pharmaceutical, industrial, and/or agricultural contexts.

In some embodiments, the aldolases and/or compositions thereof may beuseful for forming or cleaving carbon-carbon bonds.

In some embodiments, aldolases are provided that catalyze carbon-carbonbond forming reactions between an alpha-keto acid acceptor and apyruvate or a pyruvate derivative donor (see reaction scheme below). Insome embodiments, the acceptor can also be a ketone or an aldehyde. Insome embodiments, aldolases are provided that have4-hydroxy-2-oxoglutarate aldolase (such as 2-keto-4-hydroxyglutaratealdolase, 2-oxo-4-hydroxyglutarate aldolase, KHG-aldolase, EC 4.1.3.16)activity and catalyze the following reaction: 4-hydroxy-2-oxoglutarate<=> pyruvate + glyoxylate. In some embodiments, aldolases are providedthat have HMG-aldolase (such as 4-hydroxy-4-methyl-2-oxoglutaratealdolase, pyruvate aldolase,gamma-methyl-gamma-hydroxy-alpha-ketoglutaric aldolase,4-hydroxy-4-methyl-2-ketoglutarate aldolase, EC 4.1.3.17) activity andcatalyze the following reaction: 4-hydroxy-4-methyl-2-oxoglutarate <=> 2pyruvate. An HMG aldolase will also act on4-hydroxy-4-methyl-2-oxoadipate and 4-carboxy-4-hydroxy-2-oxohexadioate.

In some embodiments, aldolases, such as a pyruvate aldolase, such as,without limitation a HMG and/or a KHG aldolase, are provided thatfacilitate the production of a 3,4-substituted 2-keto-glutarate. In oneembodiment, the invention provides a method of making a 3,4-substituted2-keto-glutarate comprising: (a) providing a polypeptide having analdolase activity, such as a pyruvate aldolase activity, such as,without limitation, a HMG aldolase and/or a KMG aldolase activity; (b)providing a donor and an acceptor compound; and (c) contacting thepolypeptide of step (a) with the compounds of step (b) under conditionswherein the aldolase catalyzes the synthesis of a 3,4-substituted2-keto-glutarate, wherein optionally the donor and the acceptor are apyruvate or a pyruvate donor and an α-keto acid acceptor, a ketoneand/or an aldehyde.

In some embodiments, aldolases are provided that facilitate theproduction of R-2-hydroxy 2-(indol-3ylmethyl)-4-keto glutaric acid(R-MP), a monatin precursor. In some embodiments, a pyruvate aldolase,such as a HMG and/or a KHG aldolase, can be used in conjunction with aD-aminotransferase to make a 4-substituted D-glutamic acid or aderivative thereof. A 4-substituted D-glutamic acid and/or a derivativethereof can be used as an antibiotic, as these compounds have been foundto inhibit bacterial glutamate racemase (WO0214261A3). In oneembodiment, the invention provides a method of making a 4-substitutedD-glutamic acid comprising: (a) providing a polypeptide having analdolase activity, such as a pyruvate aldolase activity, such as,without limitation, a HMG aldolase and/or a KMG aldolase activity; (b)providing an α-keto acid acceptor and a pyruvate or a pyruvate donor;and (c) contacting the polypeptide of step (a) with the compounds ofstep (b) under conditions wherein the aldolase catalyzes the synthesisof a 4-substituted D-glutamic acid, wherein optionally the polypeptidehas pyruvate aldolase, HMG aldolase and/or KHG aldolase activity andwherein optionally the method further comprises use of aD-aminotransferase.

The invention provides isolated, synthetic or recombinant nucleic acidscomprising a nucleic acid sequence having at least about 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identityto a nucleic acid in accordance with the invention, including SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31,SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41,SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51,SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61,SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71,SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81,SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91,SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101,SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ IDNO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129,SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ IDNO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157,SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ IDNO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185,SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ IDNO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213,SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ IDNO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241,SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ IDNO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269,SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ IDNO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297,SEQ ID NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQ ID NO:305, SEQ IDNO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQID NO:317, SEQ ID NO:319, SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325,SEQ ID NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ IDNO:335, SEQ ID NO:336, SEQ ID NO:337, and SEQ ID NO:338, over a regionof at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500,1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100,2200, 2250, 2300, 2350, 2400, 2450, 2500, or more residues. In someembodiments, one or more nucleic acids encode at least one polypeptidehaving an aldolase activity, including pyruvate activity such as,without limitation, HMG and/or KHG aldolase activity. In someembodiments, the sequence identities are determined by analysis with asequence comparison algorithm or by a visual inspection.

In alternative embodiments, one or more nucleic acids encode at leastone polypeptide capable of generating an antibody that can specificallybind to a polypeptide of the invention, or, these nucleic acids can beused as probes for identifying or isolating aldolase-encoding nucleicacids, or to inhibit the expression of aldolase-expressing nucleicacids.

Nucleic acids in accordance with the invention also include isolated,synthetic or recombinant nucleic acids encoding enzymes in accordancewith the invention, such as enzymes including one or more polypeptideshaving a sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ IDNO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ IDNO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ IDNO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116,SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ IDNO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144,SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ IDNO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172,SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ IDNO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200,SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ IDNO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228,SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ IDNO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256,SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ IDNO:266, SEQ ID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:284,SEQ ID NO:286, SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:292, SEQ IDNO:294, SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ ID NO:302, SEQID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312,SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ IDNO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQID NO:332, and SEQ ID NO:334, and subsequences thereof, variants thereofand enzymatically active fragments thereof. In some embodiments, thepolypeptide has an aldolase activity, including pyruvate activity suchas, without limitation, HMG and/or KHG aldolase activity.

In some embodiments, the invention provides aldolase-encoding, such aspyruvate aldolase-, such as HMG and/or KHG aldolase-encoding nucleicacids preferably derived from mixed cultures. In some embodiments, theinvention provides carbon-carbon bond forming or cleavingenzyme-encoding nucleic acids isolated from mixed cultures comprisingpolynucleotides in accordance with the invention, such as a sequencehaving at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequenceidentity to a nucleic acid in accordance with the invention, such as SEQID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ IDNO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ IDNO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ IDNO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ IDNO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ IDNO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119,SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ IDNO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147,SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ IDNO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175,SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ IDNO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203,SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ IDNO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231,SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ IDNO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259,SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267, SEQ IDNO:269, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:285, SEQ ID NO:287,SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ IDNO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQ ID NO:305, SEQID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:315,SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321, SEQ ID NO:323, SEQ IDNO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQ ID NO:333, SEQID NO:335, SEQ ID NO:336, SEQ ID NO:337, and SEQ ID NO:338 over a regionof at least about 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, ormore.

In some embodiments, the invention provides aldolase enzyme-, such aspyruvate aldolase enzyme-, HMG and/or KHG enzyme-encoding nucleic acids,including polynucleotide sequences in accordance with the invention andthe polypeptides encoded by them, including enzymes in accordance withthe invention, such as polypeptides in accordance with the invention,such as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ IDNO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ IDNO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ IDNO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118,SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ IDNO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146,SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ IDNO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO: 162, SEQ ID NO:164, SEQID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174,SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ IDNO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202,SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ IDNO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230,SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ IDNO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258,SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQ IDNO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQ ID NO:276, SEQID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:284, SEQ ID NO:286,SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:292, SEQ ID NO:294, SEQ IDNO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ ID NO:302, SEQ ID NO:304, SEQID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314,SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ IDNO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, orSEQ ID NO:334, and enzymatically-active fragments thereof, preferablyderived from a common source, such as an environmental source. In someembodiments, the invention also provides aldolase enzyme-, such aspyruvate aldolase enzyme-, HMG and/or KHG enzyme-encoding nucleic acidspreferably derived from environmental sources, such as mixedenvironmental sources.

In some embodiments, the sequence comparison algorithm is a BLASTversion 2.2.2 algorithm where a filtering setting is set to blastall-pblastp-d “nr pataa”-F F, and all other options are set to default.

Other embodiments of the invention are isolated, synthetic orrecombinant nucleic acids including at least 10, 15, 20, 25, 30, 35, 40,45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900,1950, 2000, 2050, 2100, 2200, 2250, 2300, 2350, 2400, 2450, 2500, ormore consecutive bases of a nucleic acid sequence in accordance with theinvention, sequences substantially identical thereto, and the sequencescomplementary thereto.

In some embodiments, the isolated, synthetic or recombinant nucleicacids in accordance with the invention encodes a polypeptide having analdolase activity, including pyruvate activity such as, withoutlimitation, HMG and/or KHG aldolase activity, which is thermostable. Thethermostable polypeptide according to the invention can retain analdolase activity, such as a pyruvate aldolase activity, such as a HMGand/or a KHG aldolase activity, under conditions comprising atemperature range from about −100° C. to about −80° C., about −80° C. toabout −40° C., about −40° C. to about −20° C., about −20° C. to about 0°C., about 0° C. to about 37° C., about 0° C. to about 5° C., about 5° C.to about 15° C., about 15° C. to about 25° C., about 25° C. to about 37°C., about 37° C. to about 45° C., about 45° C. to about 55° C., about55° C. to about 70° C., about 70° C. to about 75° C., about 75° C. toabout 85° C., about 85° C. to about 90° C., about 90° C. to about 95°C., about 95° C. to about 100° C., about 100° C. to about 105° C., about105° C. to about 110° C., about 110° C. to about 120° C., or 95° C., 96°C., 97° C., 98° C., 99° C., 100° C., 101° C., 102° C., 103° C., 104° C.,105° C., 106° C., 107° C., 108° C., 109° C., 110° C., 111° C., 112° C.,113° C., 114° C., 115° C. or more. The thermostable polypeptidesaccording to the invention can retain an aldolase activity, such as apyruvate aldolase activity, such as a HMG and/or a KHG aldolaseactivity, in temperatures in the range from about −100° C. to about −80°C., about −80° C. to about −40° C., about −40° C. to about −20° C.,about −20° C. to about 0° C., about 0° C. to about 5° C., about 5° C. toabout 15° C., about 15° C. to about 25° C., about 25° C. to about 37°C., about 37° C. to about 45° C., about 45° C. to about 55° C., about55° C. to about 70° C., about 70° C. to about 75° C., about 75° C. toabout 85° C., about 85° C. to about 90° C., about 90° C. to about 95°C., about 95° C. to about 100° C., about 100° C. to about 105° C., about105° C. to about 110° C., about 110° C. to about 120° C., or 95° C., 96°C., 97° C., 98° C., 99° C., 100° C., 101° C., 102° C., 103° C., 104° C.,105° C., 106° C., 107° C., 108° C., 109° C., 110° C., 111° C., 112° C.,113° C., 114° C., 115° C. or more. In some embodiments, the thermostablepolypeptides according to the invention retains an aldolase activity ata temperature in the ranges described above, at about pH 3.0, about pH3.5, about pH 4.0, about pH 4.5, about pH 5.0, about pH 5.5, about pH6.0, about pH 6.5, about pH 7.0, about pH 7.5, about pH 8.0, about pH8.5, about pH 9.0, about pH 9.5, about pH 10.0, about pH 10.5, about pH11.0, about pH 11.5, about pH 12.0 or more.

In other embodiments, the isolated, synthetic or recombinant nucleicacids encode a polypeptide having an aldolase activity, includingpyruvate activity such as, without limitation, HMG and/or KHG aldolaseactivity, which is thermotolerant. The thermotolerant polypeptidesaccording to the invention can retain an aldolase activity, such as apyruvate aldolase activity, such as a HMG and/or a KHG aldolaseactivity, after exposure to conditions comprising a temperature in therange from about −100° C. to about −80° C., about −80° C. to about −40°C., about −40° C. to about −20° C., about −20° C. to about 0° C., about0° C. to about 5° C., about 5° C. to about 15° C., about 15° C. to about25° C., about 25° C. to about 37° C., about 37° C. to about 45° C.,about 45° C. to about 55° C., about 55° C. to about 70° C., about 70° C.to about 75° C., about 75° C. to about 85° C., about 85° C. to about 90°C., about 90° C. to about 95° C., about 95° C. to about 100° C., about100° C. to about 105° C., about 105° C. to about 110° C., about 110° C.to about 120° C., or 95° C., 96° C., 97° C., 98° C., 99° C., 100° C.,101° C., 102° C., 103° C., 104° C., 105° C., 106° C., 107° C., 108° C.,109° C., 110° C., 111° C., 112° C., 113° C., 114° C., 115° C. or more.The thermotolerant polypeptides according to the invention can retain analdolase activity, such as a pyruvate aldolase activity, such as a HMGand/or a KHG aldolase activity, after exposure to a temperature in therange from about −100° C. to about −80° C., about −80° C. to about −40°C., about −40° C. to about −20° C., about −20° C. to about 0° C., about0° C. to about 5° C., about 5° C. to about 15° C., about 15° C. to about25° C., about 25° C. to about 37° C., about 37° C. to about 45° C.,about 45° C. to about 55° C., about 55° C. to about 70° C., about 70° C.to about 75° C., about 75° C. to about 85° C., about 85° C. to about 90°C., about 90° C. to about 95° C., about 95° C. to about 100° C., about100° C. to about 105° C., about 105° C. to about 110° C., about 110° C.to about 120° C., or 95° C., 96° C., 97° C., 98° C., 99° C., 100° C.,101° C., 102° C., 103° C., 104° C., 105° C., 106° C., 107° C., 108° C.,109° C., 110° C., 111° C., 112° C., 113° C., 114° C., 115° C. or more.In some embodiments, the thermotolerant polypeptides according to theinvention retains an aldolase activity after exposure to a temperaturein the ranges described above, at about pH 3.0, about pH 3.5, about pH4.0, about pH 4.5, about pH 5.0, about pH 5.5, about pH 6.0, about pH6.5, about pH 7.0, about pH 7.5, about pH 8.0, about pH 8.5, about pH9.0, about pH 9.5, about pH 10.0, about pH 10.5, about pH 11.0, about pH11.5, about pH 12.0 or more.

The invention provides isolated, synthetic or recombinant nucleic acidscomprising a sequence that hybridizes under stringent conditions tonucleic acids in accordance with the invention, including a sequence asset forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117,SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ IDNO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145,SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ IDNO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173,SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ IDNO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201,SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ IDNO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229,SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ IDNO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257,SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ IDNO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:285,SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ IDNO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ ID NO:313,SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321, SEQ IDNO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQID NO:333, SEQ ID NO:335, SEQ ID NO:336, SEQ ID NO:337, or SEQ IDNO:338, or fragments or subsequences thereof. In some embodiments, thenucleic acids encode polypeptides having an aldolase activity, includingpyruvate activity such as, without limitation, HMG and/or KHG aldolaseactivity. The nucleic acids can be at least about 10, 15, 20, 25, 30,35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200 ormore residues in length or the full length of the gene or transcript. Insome embodiments, the stringent conditions comprise a wash stepcomprising a wash in 0.2×SSC at a temperature of about 65° C. for about15 minutes.

The invention provides nucleic acid probes for identifying or isolatingnucleic acids encoding polypeptides having an aldolase activity,including pyruvate activity such as, without limitation, HMG and/or KHGaldolase activity, wherein the probes comprise about 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000 or more, consecutive bases of a sequence in accordance with theinvention, and wherein the probes identify the nucleic acid by bindingor hybridization. The probes can comprise an oligonucleotide comprisingbetween about 10-100 consecutive bases of a sequence in accordance withthe invention, or fragments or subsequences thereof, for example, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or100 bases or more, or, any desired length in between.

The invention provides nucleic acid probes for identifying or isolatingnucleic acids encoding polypeptides having an aldolase activity,including pyruvate activity such as, without limitation, HMG and/or KHGaldolase activity, wherein the probes comprise nucleic acids comprisinga sequence at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000 or more residues of a nucleic acid in accordancewith the invention, such as a polynucleotide having at least about 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequenceidentity to a nucleic acid of the invention. In some embodiments, thesequence identities are determined by analysis with a sequencecomparison algorithm or by visual inspection. In other embodiments, theprobes can comprise an oligonucleotide comprising between at least about10-100 consecutive bases of a nucleic acid sequence in accordance withthe invention, or a subsequence thereof, for example 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 bases ormore, or, any desired length in between.

The invention provides amplification primer pairs for amplifying (suchas by PCR) a nucleic acids encoding polypeptides having aldolaseactivity, including pyruvate activity such as, without limitation, HMGand/or KHG aldolase activity, wherein each primer pair is capable ofamplifying a nucleic acid comprising a sequence in accordance with theinvention, or fragments or subsequences thereof (see the SequenceListing). One or each member of the amplification primer sequence paircan comprise an oligonucleotide comprising at least about 10 to 50, ormore, consecutive bases of the sequence, or about 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36 or more consecutive bases of the sequence. In someembodiments, the invention provides amplification primer pairs, whereineach primer pair comprises a first member having a sequence as set forthby about the first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more residuesof a nucleic acid in accordance with the invention, and a second memberhaving a sequence as set forth by about the first (the 5′) 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36 or more residues of the complementary strand of the firstmember.

The invention provides aldolase-encoding, such as pyruvatealdolase-encoding, HMG and/or KHG aldolase-encoding nucleic acidsgenerated by amplification, such as polymerase chain reaction (PCR),using an amplification primer pair in accordance with the invention. Insome embodiments, the invention provides aldolase-encoding, such aspyruvate aldolase-encoding, HMG and/or KHG aldolase-encoding nucleicacids generated by amplification, such as polymerase chain reaction(PCR), using an amplification primer pair in accordance with theinvention. In some embodiments, the invention provides methods of makingan aldolase, such as pyruvate aldolase, HMG and/or KHG aldolase enzymeby amplification, such as polymerase chain reaction (PCR), using anamplification primer pair in accordance with the invention. In someembodiments, the amplification primer pair amplifies a nucleic acid froma library, such as a gene library, such as an environmental library.

The invention provides methods of amplifying a nucleic acid encoding apolypeptide having an aldolase activity, including pyruvate activitysuch as, without limitation, HMG and/or KHG aldolase activity comprisingamplification of a template nucleic acid with an amplification primersequence pair capable of amplifying a nucleic acid sequence inaccordance with the invention, or fragments or subsequences thereof.

The invention provides expression cassettes comprising a nucleic acid inaccordance with the invention or a subsequence thereof. In someembodiments, the expression cassette can comprise the nucleic acid thatis operably linked to a promoter. The promoter can be a viral,bacterial, mammalian, fungal, yeast, or plant promoter. In someembodiments, the plant promoter can be a potato, rice, corn, wheat,tobacco or barley promoter. The promoter can be a constitutive promoter.The constitutive promoter can comprise CaMV35S. In other embodiments,the promoter can be an inducible promoter. In some embodiments, thepromoter can be a tissue-specific promoter or an environmentallyregulated or a developmentally regulated promoter. Thus, the promotercan be, such as a seed-specific, a leaf-specific, a root-specific, astem-specific or an abscission-induced promoter. In some embodiments,the expression cassette can further comprise a plant or plant virusexpression vector.

The invention provides cloning vehicles comprising an expressioncassette (such as a vector) in accordance with the invention or anucleic acid in accordance with the invention. The cloning vehicle canbe a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, abacteriophage or an artificial chromosome. The viral vector can comprisean adenovirus vector, a retroviral vector or an adeno-associated viralvector. The cloning vehicle can comprise a bacterial artificialchromosome (BAC), a plasmid, a bacteriophage P1-derived vector (PAC), ayeast artificial chromosome (YAC), or a mammalian artificial chromosome(MAC).

The invention provides transformed cells comprising nucleic acids inaccordance with the invention or expression cassettes (such as vectors)in accordance with the invention, or cloning vehicles in accordance withthe invention. In some embodiments, the transformed cell can be abacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insectcell or a plant cell. In some embodiments, the plant cell can besoybeans, rapeseed, oilseed, tomato, cane sugar, a cereal, a potato,wheat, rice, corn, tobacco or barley cell.

The invention provides transgenic non-human animals comprising a nucleicacid in accordance with the invention or an expression cassette (such asa vector) in accordance with the invention. In some embodiments, theanimal is a mouse, a rat, a pig, a goat or a sheep.

The invention provides transgenic plants comprising a nucleic acid inaccordance with the invention or an expression cassette (such as avector) in accordance with the invention. The transgenic plant can be acereal plant, a corn plant, a potato plant, a tomato plant, a wheatplant, an oilseed plant, a rapeseed plant, a soybean plant, a riceplant, a barley plant or a tobacco plant.

The invention provides transgenic seeds comprising a nucleic acid inaccordance with the invention or an expression cassette (such as avector) in accordance with the invention. The transgenic seed can be acereal plant, a corn seed, a wheat kernel, an oilseed, a rapeseed, asoybean seed, a palm kernel, a sunflower seed, a sesame seed, a peanutor a tobacco plant seed.

The invention provides antisense oligonucleotides comprising nucleicacid sequences complementary to or capable of hybridizing understringent conditions to nucleic acids in accordance with the invention.In some embodiments, the invention provides methods of inhibiting thetranslation of an aldolase, such as pyruvate aldolase, HMG and/or KHGaldolase enzyme message in a cell comprising administering to the cellor expressing in the cell an antisense oligonucleotide comprising anucleic acid sequence complementary to or capable of hybridizing understringent conditions to a nucleic acid in accordance with the invention.In some embodiments, the antisense oligonucleotide is about 10 to about50, about 20 to about 60, about 30 to about 70, about 40 to about 80, orabout 60 to about 100 bases in length, such as 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more bases inlength.

The invention provides methods of inhibiting the translation of analdolase enzyme, such as pyruvate aldolase, such as HMG and/or KHGaldolase enzyme message in a cell comprising administering to the cellor expressing in the cell an antisense oligonucleotide comprising anucleic acid sequence complementary to or capable of hybridizing understringent conditions to a nucleic acid in accordance with the invention.

The invention provides double-stranded inhibitory RNA (RNAi, or RNAinterference) molecules (including small interfering RNA, or siRNAs, forinhibiting transcription, and microRNAs, or miRNAs, for inhibitingtranslation) comprising a subsequence of a sequence in accordance withthe invention. In some embodiments, the siRNA is about 21 to about 24residues, or, about at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100 or more duplex nucleotides in length. In someembodiments, the invention provides methods of inhibiting the expressionof an aldolase enzyme, such as pyruvate aldolase, such as HMG and/or KHGaldolase enzyme in a cell comprising administering to the cell orexpressing in the cell a double-stranded inhibitory RNA (siRNA ormiRNA), wherein the RNA comprises a subsequence of a sequence inaccordance with the invention.

The invention provides isolated, synthetic or recombinant polypeptidescomprising an amino acid sequence having at least about 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identityto a polypeptide or peptide in accordance with the invention over aregion of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350 or more residues, or over the full length of the polypeptide.In some embodiments, the sequence identities are determined by analysiswith a sequence comparison algorithm or by a visual inspection.Polypeptide or peptide sequences in accordance with the inventioninclude SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ IDNO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ IDNO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ IDNO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118,SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ IDNO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146,SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ IDNO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174,SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ IDNO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202,SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ IDNO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230,SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ IDNO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258,SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQ IDNO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQ ID NO:276, SEQID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:284, SEQ ID NO:286,SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:292, SEQ ID NO:294, SEQ IDNO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ ID NO:302, SEQ ID NO:304, SEQID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314,SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ IDNO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, andSEQ ID NO:334, and subsequences thereof, variants thereof andenzymatically active fragments thereof. Polypeptides in accordance withthe invention also include fragments of at least about 10, 15, 20, 25,30, 35, 40, 45, 50, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600 or more residues in length, or over the fulllength of an enzyme. Polypeptide or peptide sequences in accordance withthe invention include sequence encoded by a nucleic acid in accordancewith the invention. Polypeptide or peptide sequences in accordance withthe invention include polypeptides or peptides specifically bound by anantibody in accordance with the invention (such as epitopes), orpolypeptides or peptides that can generate an antibody in accordancewith the invention (such as an immunogen).

In some embodiments, a polypeptide in accordance with the invention hasat least one aldolase enzyme activity, such as pyruvate aldolase, suchas HMG and/or KHG aldolase, enzyme activity. In other embodiments, apolynucleotide in accordance with the invention encodes a polypeptidethat has at least one aldolase enzyme activity, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzyme activity.

Another embodiment of the invention provides isolated, synthetic orrecombinant polypeptides or peptides comprising at least 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150 ormore consecutive bases of polypeptide or peptide sequences in accordancewith the invention, sequences substantially identical thereto, and thesequences complementary thereto. The peptide can be, such as animmunogenic fragment, a motif (such as a binding site), a signalsequence, a prepro sequence or an active site.

The invention provides isolated, synthetic or recombinant nucleic acidscomprising a sequence encoding a polypeptide having an aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase, enzyme activity anda signal sequence, wherein the nucleic acid comprises a sequence inaccordance with the invention. A “signal sequence” means a secretionsignal or other domain that facilitates secretion of the aldolase inaccordance with the invention from the host cell. The signal sequencecan be derived from another aldolase, such as pyruvate aldolase, such asHMG and/or KHG aldolase enzyme or a non-aldolase, such as non-pyruvatealdolase, such as non-HMG and/or non-KHG-aldolase enzyme (aheterologous) enzyme. In some embodiments, the invention providesisolated, synthetic or recombinant nucleic acids comprising a sequenceencoding a polypeptide having an aldolase, such as pyruvate aldolase,such as HMG and/or KHG aldolase, enzyme activity, wherein the sequencedoes not contain a signal sequence and the nucleic acid comprises asequence in accordance with the invention. In some embodiments, theinvention provides isolated, synthetic or recombinant polypeptidescomprising polypeptides in accordance with the invention lacking all orpart of a signal sequence. In some embodiments, the isolated, syntheticor recombinant polypeptide can comprise the polypeptide in accordancewith the invention comprising a heterologous signal sequence, such as aheterologous aldolase, such as pyruvate aldolase, such as HMG and/or KHGaldolase enzyme signal sequence or non-aldolase, such as non-pyruvatealdolase, such as non-HMG and/or non-KHG-aldolase enzyme signalsequence.

In some embodiments, the invention provides chimeric proteins comprisinga first domain comprising a signal sequence in accordance with theinvention and at least a second domain. The protein can be a fusionprotein. The second domain can comprise an enzyme. The protein can be anon-enzyme.

The invention provides chimeric polypeptides comprising at least a firstdomain comprising signal peptide (SP), a prepro sequence and/or acatalytic domain (CD) in accordance with the invention and at least asecond domain comprising a heterologous polypeptide or peptide, whereinthe heterologous polypeptide or peptide is not naturally associated withthe signal peptide (SP), prepro sequence and/or catalytic domain (CD).In some embodiments, the heterologous polypeptide or peptide is not analdolase, such as pyruvate aldolase, HMG and/or KHG aldolase enzyme. Theheterologous polypeptide or peptide can be amino terminal to, carboxyterminal to or on both ends of the signal peptide (SP), prepro sequenceand/or catalytic domain (CD).

The invention provides isolated, synthetic or recombinant nucleic acidsencoding a chimeric polypeptide, wherein the chimeric polypeptidecomprises at least a first domain comprising signal peptide (SP), aprepro domain and/or a catalytic domain (CD) in accordance with theinvention and at least a second domain comprising a heterologouspolypeptide or peptide, wherein the heterologous polypeptide or peptideis not naturally associated with the signal peptide (SP), prepro domainand/or catalytic domain (CD).

The invention provides isolated, synthetic or recombinant signalsequences (such as signal peptides) consisting of or comprising asequence as set forth in residues 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 40, 1 to 41, 1 to 42, 1to 43, 1 to 44, 1 to 45, 1 to 46 or 1 to 47, of a polypeptide inaccordance with the invention, such as SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ IDNO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ IDNO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ IDNO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ IDNO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124,SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ IDNO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152,SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ IDNO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190,SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ IDNO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218,SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ IDNO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246,SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ IDNO:256, SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQID NO:266, SEQ ID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:274,SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ IDNO:284, SEQ ID NO:286, SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:292, SEQID NO:294, SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ ID NO:302,SEQ ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ IDNO:312, SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQID NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330,SEQ ID NO:332, or SEQ ID NO:334. In some embodiments, the inventionprovides signal sequences comprising the first 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more aminoterminal residues of a polypeptide in accordance with the invention.

In some embodiments, the aldolase, such as pyruvate aldolase, such asHMG and/or KHG aldolase enzyme, activity comprises a specific activityfrom about 10 to about 12,000 units per milligram of protein. In otherembodiments, the aldolase, such as pyruvate aldolase, such as HMG and/orKHG aldolase, enzyme activity comprises a specific activity from about1000 to about 10,000 units per milligram of protein, or, from about 5000to about 7500 units per milligram of protein. Alternatively, thealdolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme, activity comprises a specific activity in the range from about10 to about 7500 units per milligram of protein, or, from about 5000 toabout 12,000 units per milligram of protein. In some embodiments, thealdolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme, activity comprises a specific activity in the range from about10 to about 5000 units per milligram of protein, or, from about 7500 toabout 10,000 units per milligram of protein. In order embodiments, thealdolase, such as pyruvate aldolase, such as HMG and/or KHG aldolase,enzyme activity comprises a specific activity in the range from about 10to about 2500 units per milligram of protein. Alternatively, thealdolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme, activity comprises a specific activity in the range from about10 to about 1000 units per milligram of protein. An exemplary method tomeasure the activity of different aldolases, such as pyruvate aldolases,such as HMG and/or KHG aldolase enzymes, uses a general substrate,4-carboxy-4-hydroxy-2-oxoadipate (“CHA”). A typical assay comprises 50mM sodium phosphate pH 7.5, 1 mM MgC12, 1 mM CHA, 10 μg/ml D-lactatedehydrogenase (“LDH”) from Lactobacillus leichmanii (Sigma-Aldrich, St.Louis, Mo.), 0.5 mM NADH. The assay is started by adding the enzyme tobe measured. Liberation of pyruvate, coupled to the formation of NAD+ ismonitored continuously in a spectrophotometer at 340 nm. A unit ofenzyme activity is defined as the amount that liberates sufficientpyruvate to lower the absorbance at 340 nm by 1 OD per minute.

In other embodiments, the thermotolerance of the aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzyme, comprisesretention of at least half of the specific activity of the aldolase,such as pyruvate aldolase, such as HMG and/or KHG aldolase enzyme afterbeing heated to an elevated temperature, such as a temperature fromabout 0° C. to about 20° C., about 20° C. to about 37° C., about 37° C.to about 50° C., about 50° C. to about 70° C., about 70° C. to about 75°C., about 75° C. to about 80° C., about 80° C. to about 85° C., about85° C. to about 90° C., about 90° C. to about 95° C., about 95° C. toabout 100° C., about 100° C. to about 110° C., or higher. Alternatively,the thermotolerance can comprise retention of specific activity fromabout 10 to about 12,000 units per milligram of protein, or, from about5000 to about 10,000 units per milligram of protein, after being heatedto an elevated temperature, as described above. In other embodiments,the thermotolerance can comprise retention of specific activity in therange from about 10 to about 5000 units per milligram of protein afterbeing heated to an elevated temperature, as described above.

The invention provides isolated, synthetic or recombinant polypeptidesin accordance with the invention, wherein the polypeptides comprise atleast one glycosylation site. In some embodiments, glycosylation can bean N-linked glycosylation. In some embodiments, the polypeptide can beglycosylated after being expressed in a P. pastoris or a S. pombe hostor in a mammalian host cell.

In some embodiments, the polypeptide can retain aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzyme, activityunder conditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5, pH4.0, pH 3.5, pH 3.0 or less (more acidic) pH. In other embodiments, thepolypeptide can retain an aldolase, such as pyruvate aldolase, such asHMG and/or KHG aldolase enzyme, activity under conditions comprisingabout pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5, pH11.0, pH 11.5, pH 12, pH 12.5 or more (more basic) pH. In someembodiments, the polypeptide can retain an aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzyme, activity afterexposure to conditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH4.5, pH 4.0, pH 3.5, pH 3.0 or less (more acidic) pH. In otherembodiments, the polypeptide can retain an aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzyme, activity afterexposure to conditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH9, pH 9.5, pH 10, pH 10.5, pH 11.0, pH 11.5, pH 12, pH 12.5 or more(more basic) pH.

In some embodiments, the aldolase, such as pyruvate aldolase, such asHMG and/or KHG aldolase enzyme in accordance with the invention hasactivity at under alkaline conditions, such as the alkaline conditionsof the gut, such as the small intestine. In some embodiments, thepolypeptide can retains activity after exposure to the acidic pH of thestomach.

The invention provides protein preparations comprising a polypeptide(including peptides) in accordance with the invention, wherein theprotein preparation comprises a liquid, a solid or a gel. In someembodiments, the invention provides heterodimers comprising apolypeptide in accordance with the invention and a second member, suchas a polypeptide or other (second) domain. The second member of theheterodimer can be a different aldolase, such as pyruvate aldolase, suchas HMG and/or KHG aldolase enzyme, a different enzyme or anotherprotein. In some embodiments, the second domain can be a polypeptide andthe heterodimer can be a fusion protein. In some embodiments, the seconddomain can be an epitope or a tag. In some embodiments, the inventionprovides homomultimers, including, but not limited to, homodimers,homotrimers, homotetramers, homopentamers, and homohexamers, comprisinga polypeptide in accordance with the invention.

The invention provides immobilized polypeptides (including peptides)having aldolase, such as pyruvate aldolase, such as HMG and/or KHGaldolase enzyme, activity, wherein the immobilized polypeptide comprisesa polypeptide in accordance with the invention, a polypeptide encoded bya nucleic acid in accordance with the invention, or a polypeptidecomprising a polypeptide in accordance with the invention and a seconddomain. In some embodiments, the polypeptide can be immobilized on acell, a metal, a resin, a polymer, a ceramic, a glass, a microelectrode,a graphitic particle, a bead, a gel, a plate, an array or a capillarytube.

The invention also provides arrays comprising an immobilized nucleicacid in accordance with the invention, including, such as probes inaccordance with the invention. In some embodiments, the invention alsoprovides arrays comprising an antibody in accordance with the invention.

The invention provides isolated, synthetic or recombinant antibodiesthat specifically bind to a polypeptide in accordance with the inventionor to a polypeptide encoded by a nucleic acid in accordance with theinvention. These antibodies in accordance with the invention can be amonoclonal or a polyclonal antibody. In some embodiments, the inventionprovides hybridomas comprising an antibody in accordance with theinvention, such as an antibody that specifically binds to a polypeptidein accordance with the invention or to a polypeptide encoded by anucleic acid in accordance with the invention. In some embodiments, theinvention provides nucleic acids encoding these antibodies.

The invention provides methods of isolating or identifying polypeptideshaving aldolase, such as pyruvate aldolase, such as HMG and/or KHGaldolase, enzyme activity comprising the steps of: (a) providing anantibody in accordance with the invention; (b) providing a samplecomprising polypeptides; and (c) contacting the sample of step (b) withthe antibody of step (a) under conditions wherein the antibody canspecifically bind to the polypeptide, thereby isolating or identifying apolypeptide having an aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzyme, activity.

The invention provides methods of making an anti-aldolase, such asanti-pyruvate aldolase, such as anti-HMG and/or anti-KHG aldolase enzymeantibody comprising administering to a non-human animal a nucleic acidin accordance with the invention or a polypeptide in accordance with theinvention or subsequences thereof in an amount sufficient to generate ahumoral immune response, thereby making an anti-aldolase, such asanti-pyruvate aldolase, such as anti-HMG and/or anti-KHG aldolase enzymeantibody. In some embodiments, the invention provides methods of makingan anti-aldolase, such as anti-pyruvate aldolase, such as anti-HMGand/or anti-KHG aldolase immune response (cellular or humoral)comprising administering to a non-human animal a nucleic acid inaccordance with the invention or a polypeptide in accordance with theinvention or subsequences thereof in an amount sufficient to generate animmune response (cellular or humoral).

The invention provides methods of producing a recombinant polypeptidecomprising the steps of: (a) providing a nucleic acid in accordance withthe invention operably linked to a promoter; and (b) expressing thenucleic acid of step (a) under conditions that allow expression of thepolypeptide, thereby producing a recombinant polypeptide. In someembodiments, the method can further comprise transforming a host cellwith the nucleic acid of step (a) followed by expressing the nucleicacid of step (a), thereby producing a recombinant polypeptide in atransformed cell.

The invention provides methods for identifying a polypeptide havingaldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme, activity comprising the following steps: (a) providing apolypeptide in accordance with the invention; or a polypeptide encodedby a nucleic acid in accordance with the invention; (b) providingaldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme substrate; and (c) contacting the polypeptide or a fragment orvariant thereof of step (a) with the substrate of step (b) and detectinga decrease in the amount of substrate or an increase in the amount of areaction product, wherein a decrease in the amount of the substrate oran increase in the amount of the reaction product detects a polypeptidehaving an aldolase, such as pyruvate aldolase, such as HMG and/or KHGaldolase enzyme, activity. In some embodiments, the substrate is acarbohydrate, a carbohydrate-comprising compound and/or a carbohydratemimetic.

The invention provides methods for identifying aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzyme substratecomprising the following steps: (a) providing a polypeptide inaccordance with the invention; or a polypeptide encoded by a nucleicacid in accordance with the invention; (b) providing a test substrate;and (c) contacting the polypeptide of step (a) with the test substrateof step (b) and detecting a decrease in the amount of substrate or anincrease in the amount of reaction product, wherein a decrease in theamount of the substrate or an increase in the amount of a reactionproduct identifies the test substrate as an aldolase, such as pyruvatealdolase, HMG and/or KHG aldolase enzyme substrate.

The invention provides methods of determining whether a test compoundspecifically binds to a polypeptide comprising the following steps: (a)expressing a nucleic acid or a vector comprising the nucleic acid underconditions permissive for translation of the nucleic acid to apolypeptide, wherein the nucleic acid comprises a nucleic acid inaccordance with the invention, or, providing a polypeptide in accordancewith the invention; (b) providing a test compound; (c) contacting thepolypeptide with the test compound; and (d) determining whether the testcompound of step (b) specifically binds to the polypeptide.

The invention provides methods for identifying a modulator of analdolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme, activity comprising the following steps: (a) providing apolypeptide in accordance with the invention or a polypeptide encoded bya nucleic acid in accordance with the invention; (b) providing a testcompound; (c) contacting the polypeptide of step (a) with the testcompound of step (b) and measuring an activity of the aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzyme, wherein achange in the aldolase, such as pyruvate aldolase, such as HMG and/orKHG aldolase enzyme, activity measured in the presence of the testcompound compared to the activity in the absence of the test compoundprovides a determination that the test compound modulates the aldolase,such as pyruvate aldolase, such as HMG and/or KHG aldolase enzyme,activity. In some embodiments, the aldolase, such as pyruvate aldolase,such as HMG and/or KHG aldolase enzyme, activity can be measured byproviding an aldolase, such as pyruvate aldolase, HMG and/or KHGaldolase enzyme substrate and detecting a decrease in the amount of thesubstrate or an increase in the amount of a reaction product, or, anincrease in the amount of the substrate or a decrease in the amount of areaction product. A decrease in the amount of the substrate or anincrease in the amount of the reaction product with the test compound ascompared to the amount of substrate or reaction product without the testcompound identifies the test compound as an activator of aldolase, suchas pyruvate aldolase, such as HMG and/or KHG aldolase enzyme, activity.An increase in the amount of the substrate or a decrease in the amountof the reaction product with the test compound as compared to the amountof substrate or reaction product without the test compound identifiesthe test compound as an inhibitor of aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzyme, activity.

The invention provides computer systems comprising a processor and adata storage device wherein said data storage device has stored thereona polypeptide sequence or a nucleic acid sequence in accordance with theinvention (such as a polypeptide or peptide encoded by a nucleic acid inaccordance with the invention). In some embodiments, the computer systemcan further comprise a sequence comparison algorithm and a data storagedevice having at least one reference sequence stored thereon. In otherembodiments, the sequence comparison algorithm comprises a computerprogram that indicates polymorphisms. In some embodiments, the computersystem can further comprise an identifier that identifies one or morefeatures in said sequence. In some embodiments, the invention providescomputer readable media having stored thereon a polypeptide sequence ora nucleic acid sequence in accordance with the invention. In someembodiments, the invention provides methods for identifying a feature ina sequence comprising the steps of: (a) reading the sequence using acomputer program which identifies one or more features in a sequence,wherein the sequence comprises a polypeptide sequence or a nucleic acidsequence in accordance with the invention; and (b) identifying one ormore features in the sequence with the computer program. In someembodiments, the invention provides methods for comparing a firstsequence to a second sequence comprising the steps of: (a) reading thefirst sequence and the second sequence through use of a computer programwhich compares sequences, wherein the first sequence comprises apolypeptide sequence or a nucleic acid sequence in accordance with theinvention; and (b) determining differences between the first sequenceand the second sequence with the computer program. The step ofdetermining differences between the first sequence and the secondsequence can further comprise the step of identifying polymorphisms. Insome embodiments, the method can further comprise an identifier thatidentifies one or more features in a sequence. In other embodiments, themethod can comprise reading the first sequence using a computer programand identifying one or more features in the sequence.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having an aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzyme, activity from asample, such as an environmental sample, comprising the steps of: (a)providing an amplification primer sequence pair for amplifying a nucleicacid encoding a polypeptide having an aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzyme, activity, wherein theprimer pair is capable of amplifying a nucleic acid in accordance withthe invention; (b) isolating a nucleic acid from the sample or treatingthe sample such that nucleic acid in the sample is accessible forhybridization to the amplification primer pair; and, (c) combining thenucleic acid of step (b) with the amplification primer pair of step (a)and amplifying nucleic acid from the sample, thereby isolating orrecovering a nucleic acid encoding a polypeptide having an aldolase,such as pyruvate aldolase, such as HMG and/or KHG aldolase enzyme,activity from a sample. One or each member of the amplification primersequence pair can comprise an oligonucleotide comprising anamplification primer sequence pair in accordance with the invention,such as having at least about 10 to 50 consecutive bases of a sequencein accordance with the invention. In one embodiment of the invention,the sample is an environmental sample.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having an aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzyme, activity from asample, such as an environmental sample, comprising the steps of: (a)providing a polynucleotide probe comprising a nucleic acid in accordancewith the invention or a subsequence thereof; (b) isolating a nucleicacid from the sample or treating the sample such that nucleic acid inthe sample is accessible for hybridization to a polynucleotide probe ofstep (a); (c) combining the isolated nucleic acid or the treated sampleof step (b) with the polynucleotide probe of step (a); and (d) isolatinga nucleic acid that specifically hybridizes with the polynucleotideprobe of step (a), thereby isolating or recovering a nucleic acidencoding a polypeptide having an aldolase, such as pyruvate aldolase,such as HMG and/or KHG aldolase enzyme, activity from a sample. Thesample can comprise a water sample, a liquid sample, a soil sample, anair sample or a biological sample. In some embodiments, the biologicalsample can be derived from a bacterial cell, a protozoan cell, an insectcell, a yeast cell, a plant cell, a fungal cell or a mammalian cell. Inone embodiment of the invention, the sample is an environmental sample.

The invention provides methods of generating a variant of a nucleic acidencoding a polypeptide having an aldolase, such as pyruvate aldolase,such as HMG and/or KHG aldolase enzyme, activity comprising the stepsof: (a) providing a template nucleic acid comprising a nucleic acid inaccordance with the invention; and (b) modifying, deleting or adding oneor more nucleotides in the template sequence, or a combination thereof,to generate a variant of the template nucleic acid. In some embodiments,the method can further comprise expressing the variant nucleic acid togenerate a variant aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzyme polypeptide. The modifications, additions ordeletions can be introduced by a method comprising error-prone PCR,shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexualPCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM),synthetic ligation reassembly (SLR), Chromosomal Saturation Mutagenesis(CSM) or a combination thereof. In other embodiments, the modifications,additions or deletions are introduced by a method comprisingrecombination, recursive sequence recombination, phosphothioate-modifiedDNA mutagenesis, uracil-containing template mutagenesis, gapped duplexmutagenesis, point mismatch repair mutagenesis, repair-deficient hoststrain mutagenesis, chemical mutagenesis, radiogenic mutagenesis,deletion mutagenesis, restriction-selection mutagenesis,restriction-purification mutagenesis, artificial gene synthesis,ensemble mutagenesis, chimeric nucleic acid multimer creation and acombination thereof.

In some embodiments, the method can be iteratively repeated until analdolase, such as pyruvate aldolase, HMG and/or KHG aldolase enzymehaving an altered or different activity or an altered or differentstability from that of a polypeptide encoded by the template nucleicacid is produced. In some embodiments, the variant aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzyme polypeptide isthermotolerant, and retains some activity after being exposed to anelevated temperature. In other embodiments, the variant aldolase, suchas pyruvate aldolase, such as HMG and/or KHG aldolase enzyme polypeptidehas increased glycosylation as compared to the aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzyme encoded by atemplate nucleic acid. Alternatively, the variant aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase polypeptide has analdolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme, activity under a high temperature, wherein the aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzyme encoded by thetemplate nucleic acid is not active under the high temperature. In someembodiments, the method can be iteratively repeated until an aldolase,such as pyruvate aldolase, HMG and/or KHG aldolase enzyme codingsequence having an altered codon usage from that of the template nucleicacid is produced. In other embodiments, the method can be iterativelyrepeated until an aldolase, such as pyruvate aldolase, HMG and/or KHGaldolase enzyme gene having higher or lower level of message expressionor stability from that of the template nucleic acid is produced.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having an aldolase, such as pyruvate aldolase,such as HMG and/or KHG aldolase enzyme, activity to increase itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid in accordance with the invention encoding apolypeptide having an aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzyme, activity; and, (b) identifying anon-preferred or a less preferred codon in the nucleic acid of step (a)and replacing it with a preferred or neutrally used codon encoding thesame amino acid as the replaced codon, wherein a preferred codon is acodon over-represented in coding sequences in genes in the host cell anda non-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell, thereby modifying thenucleic acid to increase its expression in a host cell.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having an aldolase, such as pyruvate aldolase,such as HMG and/or KHG aldolase enzyme, activity; the method comprisingthe following steps: (a) providing a nucleic acid in accordance with theinvention; and, (b) identifying a codon in the nucleic acid of step (a)and replacing it with a different codon encoding the same amino acid asthe replaced codon, thereby modifying codons in a nucleic acid encodingan aldolase, such as pyruvate aldolase, HMG and/or KHG aldolase enzyme.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having an aldolase, such as pyruvate aldolase,such as HMG and/or KHG aldolase enzyme, activity to increase itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid in accordance with the invention encodingan aldolase, such as pyruvate aldolase, HMG and/or KHG aldolase enzymepolypeptide; and, (b) identifying a non-preferred or a less preferredcodon in the nucleic acid of step (a) and replacing it with a preferredor neutrally used codon encoding the same amino acid as the replacedcodon, wherein a preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell, thereby modifying the nucleic acid to increaseits expression in a host cell.

The invention provides methods for modifying a codon in a nucleic acidencoding a polypeptide having an aldolase, such as pyruvate aldolase,such as HMG and/or KHG aldolase enzyme, activity to decrease itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid in accordance with the invention; and (b)identifying at least one preferred codon in the nucleic acid of step (a)and replacing it with a non-preferred or less preferred codon encodingthe same amino acid as the replaced codon, wherein a preferred codon isa codon over-represented in coding sequences in genes in a host cell anda non-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell, thereby modifying thenucleic acid to decrease its expression in a host cell. In someembodiments, the host cell can be a bacterial cell, a fungal cell, aninsect cell, a yeast cell, a plant cell or a mammalian cell.

The invention provides methods for producing a library of nucleic acidsencoding a plurality of modified aldolase, such as pyruvate aldolase,such as HMG and/or KHG aldolase enzyme active sites or substrate bindingsites, wherein the modified active sites or substrate binding sites arederived from a first nucleic acid comprising a sequence encoding a firstactive site or a first substrate binding site the method comprising thefollowing steps: (a) providing a first nucleic acid encoding a firstactive site or first substrate binding site, wherein the first nucleicacid sequence comprises a sequence that hybridizes under stringentconditions to a nucleic acid in accordance with the invention, and thenucleic acid encodes an aldolase, such as pyruvate aldolase, HMG and/orKHG aldolase enzyme active site or an aldolase, such as pyruvatealdolase, HMG and/or KHG aldolase enzyme substrate binding site; (b)providing a set of mutagenic oligonucleotides that encodenaturally-occurring amino acid variants at a plurality of targetedcodons in the first nucleic acid; and, (c) using the set of mutagenicoligonucleotides to generate a set of active site-encoding or substratebinding site-encoding variant nucleic acids encoding a range of aminoacid variations at each amino acid codon that was mutagenized, therebyproducing a library of nucleic acids encoding a plurality of modifiedaldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme active sites or substrate binding sites. In some embodiments, themethod comprises mutagenizing the first nucleic acid of step (a) by amethod comprising an optimized directed evolution system, Gene SiteSaturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR),error-prone PCR, shuffling, oligonucleotide-directed mutagenesis,assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassettemutagenesis, recursive ensemble mutagenesis, exponential ensemblemutagenesis, site-specific mutagenesis, gene reassembly, and acombination thereof. In other embodiments, the method comprisesmutagenizing the first nucleic acid of step (a) or variants by a methodcomprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

The invention provides methods for making a small molecule comprisingthe following steps: (a) providing a plurality of biosynthetic enzymescapable of synthesizing or modifying a small molecule, wherein one ofthe enzymes comprises an aldolase, such as pyruvate aldolase, HMG and/orKHG aldolase enzyme encoded by a nucleic acid in accordance with theinvention; (b) providing a substrate for at least one of the enzymes ofstep (a); and (c) reacting the substrate of step (b) with the enzymesunder conditions that facilitate a plurality of biocatalytic reactionsto generate a small molecule by a series of biocatalytic reactions. Insome embodiments, the invention provides methods for modifying a smallmolecule comprising the following steps: (a) providing an aldolase, suchas pyruvate aldolase, HMG and/or KHG aldolase enzyme, wherein the enzymecomprises a polypeptide in accordance with the invention, or, apolypeptide encoded by a nucleic acid in accordance with the invention,or a subsequence thereof; (b) providing a small molecule; and (c)reacting the enzyme of step (a) with the small molecule of step (b)under conditions that facilitate an enzymatic reaction catalyzed by thealdolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme, thereby modifying a small molecule by an aldolase, such aspyruvate aldolase, HMG and/or KHG aldolase enzymatic reaction. In someembodiments, the method can comprise a plurality of small moleculesubstrates for the enzyme of step (a), thereby generating a library ofmodified small molecules produced by at least one enzymatic reactioncatalyzed by the aldolase, such as pyruvate aldolase, such as HMG and/orKHG aldolase enzyme. In some embodiments, the method can comprise aplurality of additional enzymes under conditions that facilitate aplurality of biocatalytic reactions by the enzymes to form a library ofmodified small molecules produced by the plurality of enzymaticreactions. In other embodiments, the method can further comprise thestep of testing the library to determine if a particular modified smallmolecule that exhibits a desired activity is present within the library.The step of testing the library can further comprise the steps ofsystematically eliminating all but one of the biocatalytic reactionsused to produce a portion of the plurality of the modified smallmolecules within the library by testing the portion of the modifiedsmall molecule for the presence or absence of the particular modifiedsmall molecule with a desired activity, and identifying at least onespecific biocatalytic reaction that produces the particular modifiedsmall molecule of desired activity.

The invention provides methods for determining a functional fragment ofan aldolase, such as pyruvate aldolase, HMG and/or KHG aldolase enzymecomprising the steps of: (a) providing an aldolase, such as pyruvatealdolase, HMG and/or KHG aldolase enzyme, wherein the enzyme comprises apolypeptide in accordance with the invention, or a polypeptide encodedby a nucleic acid in accordance with the invention, or a subsequencethereof; and (b) deleting a plurality of amino acid residues from thesequence of step (a) and testing the remaining subsequence for analdolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme, activity, thereby determining a functional fragment of analdolase, such as pyruvate aldolase, HMG and/or KHG aldolase enzyme. Insome embodiments, the aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzyme, activity is measured by providing analdolase, such as pyruvate aldolase, HMG and/or KHG aldolase enzymesubstrate and detecting a decrease in the amount of the substrate or anincrease in the amount of a reaction product.

The invention provides methods for whole cell engineering of new ormodified phenotypes by using real-time metabolic flux analysis, themethod comprising the following steps: (a) making a modified cell bymodifying the genetic composition of a cell, wherein the geneticcomposition is modified by addition to the cell of a nucleic acid inaccordance with the invention; (b) culturing the modified cell togenerate a plurality of modified cells; (c) measuring at least onemetabolic parameter of the cell by monitoring the cell culture of step(b) in real time; and, (d) analyzing the data of step (c) to determineif the measured parameter differs from a comparable measurement in anunmodified cell under similar conditions, thereby identifying anengineered phenotype in the cell using real-time metabolic fluxanalysis. In some embodiments, the genetic composition of the cell canbe modified by a method comprising deletion of a sequence ormodification of a sequence in the cell, or, knocking out the expressionof a gene. In some embodiments, the method can further compriseselecting a cell comprising a newly engineered phenotype. In otherembodiments, the method can comprise culturing the selected cell,thereby generating a new cell strain comprising a newly engineeredphenotype.

The invention provides methods of increasing thermotolerance orthermostability of an aldolase, such as pyruvate aldolase, HMG and/orKHG aldolase enzyme polypeptide, the method comprising glycosylating analdolase, such as pyruvate aldolase, HMG and/or KHG aldolase enzymepolypeptide, wherein the polypeptide comprises at least thirtycontiguous amino acids of a polypeptide in accordance with theinvention; or a polypeptide encoded by a nucleic acid sequence inaccordance with the invention, thereby increasing the thermotolerance orthermostability of the aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase polypeptide. In some embodiments, the aldolase, suchas pyruvate aldolase, such as HMG and/or KHG aldolase enzyme specificactivity can be thermostable or thermotolerant at a temperature in therange from greater than about 37° C. to about 95° C.

The invention provides methods for overexpressing a recombinantaldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolasepolypeptide in a cell comprising expressing a vector comprising anucleic acid comprising a nucleic acid in accordance with the inventionor a nucleic acid sequence in accordance with the invention, wherein thesequence identities are determined by analysis with a sequencecomparison algorithm or by visual inspection, wherein overexpression iseffected by use of a high activity promoter, a dicistronic vector or bygene amplification of the vector.

The invention provides methods of making a transgenic plant comprisingthe following steps: (a) introducing a heterologous nucleic acidsequence into the cell, wherein the heterologous nucleic sequencecomprises a nucleic acid sequence in accordance with the invention,thereby producing a transformed plant cell; and (b) producing atransgenic plant from the transformed cell. In some embodiments, thestep (a) can further comprise introducing the heterologous nucleic acidsequence by electroporation or microinjection of plant cell protoplasts.In other embodiments, the step (a) can further comprise introducing theheterologous nucleic acid sequence directly to plant tissue by DNAparticle bombardment. Alternatively, the step (a) can further compriseintroducing the heterologous nucleic acid sequence into the plant cellDNA using an Agrobacterium tumefaciens host. In some embodiments, theplant cell can be a cane sugar, beet, soybean, tomato, potato, corn,rice, wheat, tobacco or barley cell.

The invention provides methods of expressing a heterologous nucleic acidsequence in a plant cell comprising the following steps: (a)transforming the plant cell with a heterologous nucleic acid sequenceoperably linked to a promoter, wherein the heterologous nucleic sequencecomprises a nucleic acid in accordance with the invention; (b) growingthe plant under conditions wherein the heterologous nucleic acidssequence is expressed in the plant cell. In some embodiments, theinvention provides methods of expressing a heterologous nucleic acidsequence in a plant cell comprising the following steps: (a)transforming the plant cell with a heterologous nucleic acid sequenceoperably linked to a promoter, wherein the heterologous nucleic sequencecomprises a sequence in accordance with the invention; (b) growing theplant under conditions wherein the heterologous nucleic acids sequenceis expressed in the plant cell.

The invention provides feeds or foods comprising a polypeptide inaccordance with the invention, or a polypeptide encoded by a nucleicacid in accordance with the invention. In some embodiments, theinvention provides foods, feeds, liquids, such as beverages (such asfruit juices or beer), breads or doughs or bread products, or beverageprecursors (such as wort), comprising a polypeptide in accordance withthe invention. In other embodiments, the invention provides foods,feeds, or beverage additives comprising a polypeptide in accordance withthe invention. In some embodiments, the invention provides foods ornutritional supplements, such as for a human or an animal, comprising apolypeptide in accordance with the invention, such as a polypeptideencoded by the nucleic acid in accordance with the invention.

In some embodiments, the polypeptide in the food or nutritionalsupplement can be glycosylated. In some embodiments, the inventionprovides edible enzyme delivery matrices comprising a polypeptide inaccordance with the invention, such as a polypeptide encoded by thenucleic acid in accordance with the invention. In some embodiments, thedelivery matrix comprises a pellet. In some embodiments, the polypeptidecan be glycosylated. In some embodiments, the aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzyme, activity isthermotolerant. In other embodiments, the aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzyme, activity isthermostable.

The invention provides foods, feeds or nutritional supplementscomprising a polypeptide in accordance with the invention. In someembodiments, the invention provides methods for utilizing an aldolase,such as pyruvate aldolase, HMG and/or KHG aldolase enzyme as anutritional supplement in an animal diet, the method comprising:preparing a nutritional supplement containing an aldolase, such aspyruvate aldolase, HMG and/or KHG aldolase enzyme comprising at leastthirty contiguous amino acids of a polypeptide in accordance with theinvention; and administering the nutritional supplement to an animal.The animal can be a human, a ruminant or a monogastric animal. Thealdolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme can be prepared by expression of a polynucleotide encoding thealdolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme in an organism selected from the group consisting of a bacterium,a yeast, a plant, an insect, a fungus and an animal. The organism can beselected from the group consisting of an S. pombe, S. cerevisiae, Pichiapastoris, E. coli, Streptomyces sp., Bacillus sp. Pseudomonas sp.,Aspergillus sp. and Lactobacillus sp.

The invention provides edible enzyme delivery matrices comprising athermostable recombinant aldolase, such as pyruvate aldolase, such asHMG and/or KHG aldolase enzyme, such as a polypeptide in accordance withthe invention. In some embodiments, the invention provides methods fordelivering an aldolase, such as pyruvate aldolase, HMG and/or KHGaldolase enzyme supplement to an animal, the method comprising:preparing an edible enzyme delivery matrix in the form of pelletscomprising a granulate edible carrier and a thermostable recombinantaldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme, wherein the pellets readily disperse the aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzyme containedtherein into aqueous media, and administering the edible enzyme deliverymatrix to the animal. The recombinant aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzyme can comprise apolypeptide in accordance with the invention. The aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzyme can beglycosylated to provide thermostability at pelletizing conditions. Thedelivery matrix can be formed by pelletizing a mixture comprising agrain germ and an aldolase, such as pyruvate aldolase, HMG and/or KHGaldolase enzyme. The pelletizing conditions can include application ofsteam. The pelletizing conditions can comprise application of atemperature in excess of about 80° C. for about 5 minutes and the enzymeretains a specific activity of at least 350 to about 900 units permilligram of enzyme.

In some embodiments, the invention provides pharmaceutical compositionscomprising an aldolase, such as pyruvate aldolase, HMG and/or KHGaldolase enzyme in accordance with the invention, or a polypeptideencoded by a nucleic acid in accordance with the invention. In someembodiments, the pharmaceutical composition acts as a digestive aid.

In some embodiments, a carbon-carbon bond-containing compound iscontacted a polypeptide in accordance with the invention having aldolaseenzyme activity, such as pyruvate aldolase, such as HMG and/or KHGaldolase enzyme activity, at a pH ranging from about pH 3.0 to about9.0, about 3.0 to about 10.0, about 3.0 to about 11.0 or more. In otherembodiments, a carbon-carbon bond-containing compound is contacted withthe aldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme, at a temperature of at least about 55° C., 60° C., 65° C., 70°C., 75° C., 80° C., 85° C., 90° C., or more.

This disclosure provides, among other things, polypeptides that areuseful in facilitating a reaction in processes for producing monatin,monatin derivatives, and salts thereof, for example in the production ofR-2-hydroxy 2-(indol-3ylmethyl)-4-keto glutaric acid (also referred toas R-alpha keto acid monatin, R-monatin precursor, R-MP, and the alphaketo form of monatin), a precursor for certain stereoisomers of monatin,such as R,R and S,R monatin. The disclosure also provides methods ofmaking monatin, monatin derivatives, and salts and internal condensationproducts thereof using one or more polypeptides of the invention. Themethods of synthesizing R-MP, stereoisomers of monatin and/orstereoisomers of monatin derivatives include the use of one or morepolypeptides with aldolase activity of any of SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ IDNO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ IDNO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ IDNO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ IDNO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124,SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ IDNO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152,SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ IDNO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180,SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ IDNO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208,SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ IDNO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236,SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ IDNO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQID NO:256, SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264,SEQ ID NO:266, SEQ ID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ IDNO:274, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQID NO:284, SEQ ID NO:286, SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:292,SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ IDNO:302, SEQ ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320,SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ IDNO:330, SEQ ID NO:332, and SEQ ID NO:334, and enzymatically activefragments thereof.

Also, the methods of synthesizing R-MP, stereoisomers of monatin and/orstereoisomers of monatin derivatives may include the use of apolypeptide with aldolase activity encoded by a nucleic acid sequencehaving at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, orcomplete (100%) sequence identity to nucleic acid in accordance with theinvention, including SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117,SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ IDNO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145,SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ IDNO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173,SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ IDNO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201,SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ IDNO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229,SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ IDNO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257,SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ IDNO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:285,SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ IDNO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ ID NO:313,SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321, SEQ IDNO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQID NO:333, SEQ ID NO:335, SEQ ID NO:336, SEQ ID NO:337, and SEQ IDNO:338 over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45,50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950,2000, 2050, 2100, 2200, 2250, 2300, 2350, 2400, 2450, 2500, or moreresidues.

Furthermore, the methods of synthesizing R-MP, stereoisomers of monatinand/or stereoisomers of monatin derivatives may include the use of apolypeptide with aldolase activity encoded by a nucleic acid sequencethat hybridizes under stringent condition to a nucleic acid of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31,SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41,SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51,SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61,SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71,SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81,SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91,SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101,SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ IDNO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129,SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ IDNO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157,SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ IDNO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185,SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ IDNO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213,SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ IDNO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241,SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ IDNO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269,SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ IDNO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297,SEQ ID NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQ ID NO:305, SEQ IDNO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQID NO:317, SEQ ID NO:319, SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325,SEQ ID NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ IDNO:335, SEQ ID NO:336, SEQ ID NO:337, and SEQ ID NO:338.

The invention provides a method, comprising: producing a product chosenfrom monatin, monatin derivatives, salts thereof, and combinationsthereof in a multi-step pathway, wherein a reaction in the pathway isfacilitated by one or more polypeptides chosen from isolated orrecombinant polypeptides comprising the amino acid sequence of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52,SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:64,SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74,SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84,SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94,SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:106,SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ IDNO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134,SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ IDNO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162,SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ IDNO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190,SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ IDNO:200, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220,SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ IDNO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248,SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ IDNO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQ ID NO:276,SEQ ID NO:278, SEQ ID NO:282, SEQ ID NO:284, SEQ ID NO:286, SEQ IDNO:288, SEQ ID NO:290, SEQ ID NO:292, SEQ ID NO:294, SEQ ID NO:296, SEQID NO:298, SEQ ID NO:300, SEQ ID NO:302, SEQ ID NO:304, SEQ ID NO:306,SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ IDNO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, or SEQ IDNO:334, or fragments or subsequences thereof having aldolase activity.In some embodiments, the fragments or subsequences thereof have analdolase activity of at least 0.2 mg MP/mg protein/hr. In otherembodiments, the fragments or subsequences thereof have an aldolaseactivity of at least 0.1 mg MP/mg protein/hr. In some embodiments, thereaction facilitated by one or more polypeptides in accordance with theinvention is performed in about 1.0 to about 10.0 mM MgCl₂. In otherembodiments, the reaction facilitated by one or more polypeptides inaccordance with the invention is performed at about pH 7.0 to about pH11.5. In still other embodiments, the reaction facilitated by one ormore polypeptides in accordance with the invention is performed in about0.005% to about 1% polysorbate detergent.

In some embodiments, the reaction is a reaction betweenindole-3-pyruvate and a C3 carbon source. In some embodiments, thereaction preferentially producesR-2-hydroxy-2-(indol-3-yl-methyl)-4-ketoglutaric acid overS-2-hydroxy-2-(indol-3-yl-methyl)-4-ketoglutaric acid.

In some embodiments, the product made by the multi-step pathway ismonatin, salts thereof and combinations thereof.

In other embodiments, at least one of R,R-monatin, R—S monatin, or acombination thereof is produced in greater quantity than eitherS,S-monatin or S,R-monatin in the multi-step pathway. In someembodiments, R,R monatin is produced in greater quantity thanR,S-monatin, S,S-monatin and S,R-monatin in the multi-step pathway.

The invention provides a method, comprising: producing a product chosenfrom monatin, monatin derivatives, salts thereof, and combinationsthereof in a multi-step pathway, wherein a reaction in the pathway isfacilitated by at least one polypeptide encoded by a nucleic acidsequence that comprises a sequence having a percent sequence identity ofat least 50% to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ IDNO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ IDNO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ IDNO:97, SEQ ID NO:99, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ IDNO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127,SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ IDNO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155,SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ IDNO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183,SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ IDNO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:203, SEQID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213,SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ IDNO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241,SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ IDNO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269,SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ IDNO:281, SEQ ID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQ ID NO:289, SEQID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299,SEQ ID NO:301, SEQ ID NO:303, SEQ ID NO:305, SEQ ID NO:307, SEQ IDNO:309, SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQID NO:319, SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327,SEQ ID NO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ ID NO:335, SEQ IDNO:336, SEQ ID NO:337, or SEQ ID NO:338. In some embodiments, thepercent sequence identity is at least 95%. In other embodiments, thepercent sequence identity is 100%.

The invention provides a method comprising a reaction thatpreferentially produces R-2-hydroxy-2-(indol-3-yl-methyl)-4-ketoglutaricacid over S-2-hydroxy-2-(indol-3-yl-methyl)-4-ketoglutaric acid whereinat least one polypeptide encoded by a nucleic acid sequence thatcomprises a sequence having at least 95% sequence identity to SEQ ID NO:28, SEQ ID NO:116, SEQ ID NO:298, SEQ ID NO: 44, SEQ ID NO:54, SEQ IDNO: 148, SEQ ID NO: 46, SEQ ID NO:134, SEQ ID NO:142, SEQ ID NO:122, SEQID NO:74, SEQ ID NO: 64, SEQ ID NO: 108, SEQ ID NO:96, SEQ ID NO:126,SEQ ID NO:80, SEQ ID NO:36, SEQ ID NO:62, SEQ ID NO:112, SEQ ID NO:130,SEQ ID NO:94, SEQ ID NO:58, SEQ ID NO:50, SEQ ID NO:106, SEQ ID NO:42,SEQ ID NO:278, SEQ ID NO:162, SEQ ID NO:276, SEQ ID NO:178, SEQ IDNO:166, SEQ ID NO:218, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:244, SEQID NO:250, SEQ ID NO:252, SEQ ID NO:264, SEQ ID NO:268, SEQ ID NO:272,SEQ ID NO:184, SEQ ID NO:282, SEQ ID NO:186, SEQ ID NO:192, SEQ IDNO:200, SEQ ID NO:284, SEQ ID NO:172, SEQ ID NO:180, SEQ ID NO:168, SEQID NO:228, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, and SEQ IDNO:156 is utilized to faciliate one reaction in a multi-step pathway.

The invention provides a method comprising: producing a product chosenfrom monatin, monatin derivatives, salts thereof, and combinationsthereof in a multi-step pathway, wherein a reaction in the pathway isfacilitated by at least one polypeptide encoded by a nucleic acidsequence that comprises a sequence that hybridizes under stringentcondition to a nucleic acid of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119,SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ IDNO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147,SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ IDNO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175,SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ IDNO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:203, SEQ ID NO:205,SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ IDNO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233,SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ IDNO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261,SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269, SEQ IDNO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:281, SEQID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291,SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ IDNO:301, SEQ ID NO:303, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319,SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ IDNO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ ID NO:335, SEQ ID NO:336, SEQID NO:337, or SEQ ID NO:338.

The invention also provides a method comprising: producing a productchosen from monatin precursor, salts thereof, and combinations thereof,in a multi-step pathway, wherein a reaction in the pathway isfacilitated by one or more polypeptides chosen from isolated orrecombinant polypeptides comprising the amino acid sequence of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52,SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:64,SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74,SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84,SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94,SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:104, SEQ ID NO:106,SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ IDNO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134,SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ IDNO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162,SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ IDNO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190,SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ IDNO:200, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220,SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ IDNO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248,SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ IDNO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQ ID NO:276,SEQ ID NO:278, SEQ ID NO:282, SEQ ID NO:284, SEQ ID NO:286, SEQ IDNO:288, SEQ ID NO:290, SEQ ID NO:292, SEQ ID NO:294, SEQ ID NO:296, SEQID NO:298, SEQ ID NO:300, SEQ ID NO:302, SEQ ID NO:304, SEQ ID NO:306,SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ IDNO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, or SEQ IDNO:334, or fragments or subsequences thereof having aldolase activity,wherein said monatin precursor, salts thereof, and combinations thereofis sweet.

The invention additionally provides a method comprising: producing aproduct chosen from monatin precursor, salts thereof, and combinationsthereof, in a multi-step pathway, wherein a reaction in the pathway isfacilitated by at least one polypeptide encoded by a nucleic acidsequence that comprises a sequence having a percent sequence identity ofat least 50% to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ IDNO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ IDNO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ IDNO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ IDNO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121,SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ IDNO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149,SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ IDNO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177,SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ IDNO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQID NO:197, SEQ ID NO:199, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207,SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ IDNO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235,SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ IDNO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263,SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ IDNO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:281, SEQ ID NO:283, SEQID NO:285, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293,SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ IDNO:303, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321,SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ IDNO:331, SEQ ID NO:333, SEQ ID NO:335, SEQ ID NO:336, SEQ ID NO:337, orSEQ ID NO:338, wherein said monatin precursor, salts thereof, andcombinations thereof is sweet.

The invention further provides a method, comprising: producing a productchosen from monatin precursor, salts thereof, and combinations thereof,in a multi-step pathway, wherein a reaction in the pathway isfacilitated by at least one polypeptide encoded by a nucleic acidsequence that comprises a sequence that hybridizes under stringentcondition to a nucleic acid of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119,SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ IDNO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147,SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ IDNO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175,SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ IDNO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:203, SEQ ID NO:205,SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ IDNO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233,SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ IDNO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261,SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269, SEQ IDNO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:281, SEQID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291,SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ IDNO:301, SEQ ID NO:303, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319,SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ IDNO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ ID NO:335, SEQ ID NO:336, SEQID NO:337, or SEQ ID NO:338, wherein said monatin precursor, saltsthereof, and combinations thereof is sweet.

In an effort to be concise, where ever intermediates/products areidentified in the specification and claims (such as monatin, monatinprecursor, or monatin derivative(s)) as being formed, the term “and/orsalts thereof” should be understood to be included where applicable. Inother words, for example, the phrase “indole-3-pyruvate is converted toMP” should be understood to read “indole-3-pyruvic acid is converted toMP and/or salts thereof.” A person of ordinary skill, in fact, wouldappreciate that under reaction conditions shown the salts of theintermediates/products are in fact present.

According to some embodiments, the method produces a monatin or monatinderivative composition wherein the monatin or monatin derivativecomponent of the composition includes only the R,R and S,R forms ofmonatin or monatin derivative. The term “only” when used to indicatethat only certain isomers are formed, means that the pathway wouldproduce only the identified isomers if racemization did not occur.Consequently, the term “only” should not be taken to mean absence ofother isomers, but rather a person of ordinary skill would understandthat other isomeric forms may be present in a relatively small amountdue to racemization which may occur. According to some embodiments, themethod produces a composition wherein the monatin or monatin derivativecomponent of the composition includes only the R,R form of monatin ormonatin derivative (except to the extent racemization occurs resultingin other isomeric forms).

As used herein, the phrase “monatin composition” means compositionsincluding one or more isomers of monatin; the term can also mean only asingle isomeric form of monatin and nothing else.

As used herein, the phrase “monatin derivative composition” meanscompositions including one or more isomers of a monatin derivative; theterm can also mean only a single isomeric form of the monatin derivativeand nothing else.

As used herein, the phrase “monatin derivative” has the followingstructure:

wherein, R_(a), R_(b), R_(c), R_(d), and R_(e) each independentlyrepresent any substituent selected from a hydrogen atom, a hydroxylgroup, a C₁-C₃ alkyl group, a C₁-C₃ alkoxy group, an amino group, or ahalogen atom, such as an iodine atom, bromine atom, chlorine atom, orfluorine atom. However, R_(a), R_(b), R_(c), R_(d), and R_(e) cannotsimultaneously all be hydrogen. Alternatively, R_(b) and R_(c), and/orR_(d) and R_(e) may together form a C₁-C₄ alkylene group, respectively.

As used herein, “substituted indole-3-pyruvate” means one or more carbonatoms of the indole ring of the indole-3-pyruvate is independentlysubstituted with one or more of the R_(a), R_(b), R_(c), R_(d), andR_(e) substituent groups defined above. However, R_(a), R_(b), R_(c),R_(d), and R_(e) cannot simultaneously all be hydrogen. Alternatively,R_(b) and R_(c), and/or R_(d) and R_(e) may together form a C₁-C₄alkylene group, respectively.

As used herein, “substituted tryptophan” means one or more carbon atomsof the indole ring of the tryptophan is independently substituted withone or more of the R_(a), R_(b), R_(c), R_(d), and R_(e) substituentgroups defined above. However, R_(a), R_(b), R_(c), R_(d), and R_(e)cannot simultaneously all be hydrogen. Alternatively, R_(b) and R_(c),and/or R_(d) and R_(e) may together form a C₁-C₄ alkylene group,respectively. In one embodiment, the substituted tryptophan contains thesame substituent group(s) on the indole ring as the final monatinderivative.

Furthermore, the biosynthetic pathways for producing monatin describedherein can utilize a substituted tryptophan to yield monatin derivativesthat are likely to be sweet. In some embodiments, the substitutedtryptophan to be used in the biosynthetic pathways described hereininclude chlorinated tryptophan and 5-hydroxytryptophan.

For example, chlorinated D-tryptophans, which have structuralsimilarities to R,R monatin, have been identified as non-nutritivesweeteners (particularly 6-chloro-D-tryptophan). Similarly, halogenatedand hydroxy-substituted forms of monatin have been found to be sweet.U.S. Published Patent Application No. 2005/0118317. Halogens andhydroxyl groups could be substitutable for hydrogen, particularly onpositions 1-4 of the benzene ring in the indole of tryptophan, withoutinterfering in subsequent conversions to D- or L-tryptophan,indole-3-pyruvate, MP, or monatin. Substituted indoles have been shownin the literature to be suitable substrates for PLP-enzymes and haveyielded substituted tryptophans. Fukuda, D. S., et al., “Production ofSubstituted L-Tryptophans by Fermentation,” Appl. Environ. Microbiol.,21:841-43 (1971). The halogen does not appear to sterically hinder thetryptophan synthase beta subunits catalytic mechanism and theenantiospecificity was also intact.

In some embodiments of the present invention, a process for producing amonatin composition is provided, which includes producingindole-3-pyruvate from L-tryptophan, producing 2-hydroxy2-(indol-3ylmethyl)-4-keto glutaric acid (“monatin precursor” or “MP”)from indole-3-pyruvate, and producing monatin from MP. The reaction ofL-tryptophan to produce indole-3-pyruvate is facilitated by an enzymehaving greater specificity, greater activity, or both for L-tryptophanthan for R-MP, R,R monatin, or both. According to certain embodiments,the reaction of indole-3-pyruvate is facilitated by an enzyme havingR-specific aldolase activity and consequently produces R-MP. Accordingto certain embodiments, a racemase enzyme is provided which canfacilitate the epimerization of the amino acid byproduct of thetryptophan reaction from one isomeric form to another isomeric form.

In some embodiments according to the invention, a process for producinga monatin composition is provided, which includes producingindole-3-pyruvate from L-tryptophan, producing 2-hydroxy2-(indol-3ylmethyl)-4-keto glutaric acid (“monatin precursor” or “MP”)from indole-3-pyruvate, and producing monatin from MP. The reaction ofL-tryptophan to produce indole-3-pyruvate is facilitated by an enzymehaving greater specificity, greater activity, or both for L-tryptophanthan for R-MP, R,R monatin, or both, and the reaction of MP to formmonatin is facilitated by an enzyme, which is stereoselective for R-MP.The term “stereoselective” means that an enzyme has greater specificity,greater activity, or both for one isomer—in this case for R-MP versusS-MP—over another. In preferred embodiments, a stereoselective enzymehas limited activity for one isomer as compared to another. “Limited”activity means activity that is minimally or not perceptible, forexample as determined according to experiments provided herein.

It should be noted that, where references are made to a series ofreactions such as in the preceding paragraphs, the invention does notrequire each step to be explicitly performed; it is sufficient that thesteps may be implicitly performed. In other words, for example, theprocess for producing a monatin composition, which includes producingindole-3-pyruvate from L-tryptophan, producing 2-hydroxy2-(indol-3ylmethyl)-4-keto glutaric acid (“monatin precursor” or “MP”)from indole-3-pyruvate, and producing monatin from MP, wherein eachreaction is facilitated by an appropriate enzyme, can be performed bycombining L-tryptophan with the enzymes and setting conditions so thatthe enumerated reactions could occur. In such an instance L-tryptophancould react to produce indole-3-pyruvate, the indole-3-pyruvate producedfrom the L-tryptophan reaction could react to form MP, and the MPproduced from the indole-3-pyruvate reaction could react to formmonatin. The process could also be performed, by way of example, byproviding a compound that can produce L-tryptophan, under conditionssuitable for L-tryptophan production to occur and combining thatcompound with enzymes capable of facilitating the series of reactionsset forth under conditions which would be suitable for those reactionsto occur. As yet another example, the process could be performed byproviding a microorganism genetically engineered to produce monatinaccording to the described pathway, and providing appropriate conditionsfor the fermentation process to occur. For example, a microorganism,which naturally produces large amounts of L-tryptophan could begenetically engineered to produce or over-produce one or more of theenzymes used to facilitate reactions in the pathway to monatin, andappropriate conditions could be provided so that the microorganism wouldthereby produce monatin.

In other embodiments according to the invention, a process for producingmonatin is provided, in which a substrate forms an L-amino acid whenL-tryptophan is converted to indole-3-pyruvate, indole-3-pyruvate reactsto form MP (which can include both R-MP and S-MP though preferablyincludes only or predominately R-MP), and the L-amino acid reacts toregenerate (also referred to as “recycle”) the substrate when R-MP isconverted to R,R monatin. The reaction of R-MP to form R,R monatin isfacilitated by a stereoinverting aminotransferase such as D-methionineaminotransferase (EC 2.6.1.41) or an enzyme having D-phenylglycineaminotransferase activity.

In other embodiments according to the invention, a process for producinga monatin composition is provided, which includes producing D-tryptophanfrom L-tryptophan, producing indole-3-pyruvate from D-tryptophan,producing R-MP from indole-3-pyruvate, and producing R,R monatin fromR-MP. The production of the D-tryptophan from the L-tryptophan isfacilitated by a tryptophan racemase and functional equivalents thereof.In certain further embodiments, the reactions of D-tryptophan to formindole-3-pyruvate and of MP to form monatin are facilitated by the sameenzyme. In yet other further embodiments, the reaction ofindole-3-pyruvate is facilitated by an enzyme having R-specific aldolaseactivity and consequently R-MP is formed, and the reactions ofD-tryptophan to form indole-3-pyruvate and of R-MP to form R,R monatinare facilitated by the same enzyme.

In some embodiments according to the invention, a process for producinga monatin derivative is provided, which includes producing the monatinderivative from a substituted indole-3-pyruvate and pyruvate, using anenzyme having R-specific aldolase activity to catalyze the reaction.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages in accordance with the invention will beapparent from the description and drawings, and from the claims. Asshould be realized from the description herein, the invention is capableof modifications in various embodiments, all without departing from thespirit and scope of the present invention. Accordingly, the drawings anddetailed description are to be regarded as illustrative in nature andnot restrictive.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

BRIEF DESCRIPTION OF DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIG. 1 is a flow chart that shows an example of an enzymatic process forproducing R,R monatin from L-tryptophan in accordance with theinvention. In this example, the process includes using anL-aminotransferase (examples of which include an L-tryptophanaminotransferase, an L-aromatic aminotransferase, an L-aspartateaminotransferase, and an L-alanine aminotransferase) in the reaction ofL-tryptophan that has greater specificity and/or selectivity forL-tryptophan as a substrate than for R-MP and/or the process includesusing an L-amino acid oxidase with limited activity and/or specificityfor R,R monatin as a substrate. In the specific example diagrammed inFIG. 1, an L-aminotransferase or L-amino acid oxidase convertsL-tryptophan to indole-3-pyruvate, indole-3-pyruvate is reacted with anR-specific aldolase and pyruvate to produce R-alpha-keto acid monatin(R-MP), and R-MP is converted to R,R monatin by a D-aminotransferase ora D-amino acid dehydrogenase. As shown on FIG. 1, the reactions arereversible, but for the purposes of the invention, it is not requiredthat the reactions proceed in the reverse direction.

FIG. 2 is a flow chart that shows an example of another process forproducing R,R monatin in accordance with the invention. In this example,the process includes using an enzyme to convert R-MP to monatin which isstereoselective for R-MP. In the specific example diagrammed in FIG. 2,tryptophan is shown to be converted to indole-3-pyruvate in a reversiblereaction. The indole-3-pyruvate can be reacted with a non-stereospecificaldolase to reversibly form alpha-keto acid monatin (both R- and S-MP).The R-MP is reversibly converted to R,R monatin by a stereoselectiveD-aminotransferase or a stereoselective D-amino acid dehydrogenase. AnyS-MP that is formed by the non-stereospecific aldolase can be convertedback into indole-3-pyruvate if a stereoselective D-aminotransferase orD-amino acid dehydrogenase is utilized. For the purposes of theinvention, it is not required that the reactions shown as beingreversible proceed in the reverse direction.

FIG. 3 is a flow chart that shows an example of yet another process forproducing R,R monatin from L-tryptophan in accordance with the inventionIn this example, the process includes converting L-tryptophan toD-tryptophan using a tryptophan racemase and using a D-amino acidproduct in the reaction coupled to the reaction formingindole-3-pyruvate as a substrate in the reaction coupled to the reactionforming R,R monatin. In the specific example diagrammed in FIG. 3,L-tryptophan is converted to D-tryptophan by a tryptophan racemase in areversible reaction. The D-tryptophan is reacted withalpha-ketoglutarate (α-KG) and a broad specificity D-aminotransferase toproduce indole-3-pyruvate and D-glutamate. Indole-3-pyruvate is reactedwith pyruvate and an R-specific aldolase and converted to R-alpha-ketoacid monatin (R-MP), and R-MP is reacted with a broad specificityD-aminotransferase and D-glutamate to form R,R monatin andalpha-ketoglutarate (α-KG). As shown on FIG. 3, each of the reactionsare reversible, but for the purposes of the invention, it is notrequired that the reactions proceed in the reverse direction.

FIG. 4 is a flow chart that shows an example of yet another process forproducing R,R monatin from L-tryptophan in accordance with theinvention. In this example, the process includes converting the L-aminoacid formed in the reaction coupled with the L-tryptophan reaction to aD-amino acid; this D-amino acid acts as an amino donor for the reactionin which R-MP is converted to R,R monatin. In the specific examplediagrammed in FIG. 4, L-tryptophan is reacted with an L-aminotransferaseand alpha-ketoglutarate to produce indole-3-pyruvate and L-glutamate.Indole-3-pyruvate is reacted with pyruvate and an R-specific aldolaseand converted to R-alpha-keto acid monatin (R-MP), and R-MP is reactedwith a broad specificity D-aminotransferase and D-glutamate to form R,Rmonatin and alpha-ketoglutarate. As shown on FIG. 4, the reactions arereversible, but for the purposes of the invention, it is not requiredthat the reactions proceed in the reverse direction.

FIG. 5 is a flow chart that shows an example of yet another process forproducing R,R monatin from L-tryptophan in accordance with theinvention. In this example, the process includes enzymaticallyfacilitating the conversion of R-MP to R,R monatin using astereoinverting enzyme so that the L-amino acid formed by the reactioncoupled to the L-tryptophan reaction can be used as a substrate for thereaction coupled to the R-MP to R,R monatin reaction. In the specificexample diagrammed in FIG. 5, L-tryptophan is reacted with anL-aminotransferase and oxaloacetate, pyruvate or alpha-ketoglutarate(α-KG) to produce indole-3-pyruvate, and L-aspartate (if oxaloacetate isused), L-alanine (if pyruvate is used) or L-glutamate (if α-KG is used).Indole-3-pyruvate is reacted with pyruvate and an R-specific aldolaseand converted to R-alpha-keto acid monatin (R-MP), and R-MP is reactedwith a stereoinverting aminotransferase and L-aspartate, L-alanine orL-glutamate to form R,R monatin and oxaloacetate (if L-aspartate isused), pyruvate (if L-alanine is used) or alpha-ketoglutarate (α-KG, ifL-glutamate is used). As shown on FIG. 5, the reactions are reversible,but for the purposes of the invention, it is not required that thereactions proceed in the reverse direction.

FIG. 6 is a flow chart that shows an example of yet another process forproducing R,R monatin in accordance with the present invention. In thisexample, the process includes recycling the L-amino acid produced in thereaction forming indole-3-pyruvate with the D-amino acid used as areactant with R-MP in the reaction forming R,R monatin through a seriesof conversion reactions. In the specific example diagrammed in FIG. 6,L-tryptophan is reversibly reacted with an L-aminotransferase andoxaloacetate to produce indole-3-pyruvate and L-aspartate.Indole-3-pyruvate is reacted in a reversible manner with pyruvate and anR-specific aldolase and converted to R-alpha-keto acid monatin (R-MP),and R-MP is reversibly reacted with a D-aminotransferase and D-alanineto form R,R monatin and pyruvate. The L-aspartate is converted toL-alanine and CO₂ using an aspartate 4-decarboxylase. The L-alanine isconverted to D-alanine with an alanine racemase. For the purposes of theinvention, it is not required that the reactions shown as beingreversible proceed in the reverse direction.

FIG. 7 is a flow chart that shows an example of yet another process forproducing R,R monatin in accordance with the present invention. In thisexample, the process includes pushing the L-tryptophan reaction forward(i.e., driving the reaction toward the production of indole-3-pyruvate)by converting the L-amino acid byproduct of that reaction into anotherproduct. In this example, the L-amino acid L-aspartate byproduct isconverted into L-alanine in an irreversible reaction using adecarboxylase. In the specific example diagrammed in FIG. 7,L-tryptophan is reversibly reacted with an L-aminotransferase and withalpha-ketoglutarate (α-KG) or oxaloacetate to produce indole-3-pyruvateand L-glutamate (if α-KG is used) or L-aspartate (if oxaloacetate isused). Indole-3-pyruvate is reversibly reacted with pyruvate and anR-specific aldolase and converted to R-alpha-keto acid monatin (R-MP).R-MP is reacted in a reversible manner with a D-aminotransferase and aD-amino acid to form R,R monatin and any of oxaloacetate, pyruvate orα-KG. The L-glutamate or L-aspartate that was a product of theL-aminotransferase reaction is converted to either 4-aminobutanoate andCO₂ (if L-glutamate is the substrate) or to β-alanine and CO₂ (ifL-aspartate is the substrate) using a glutamic acid or an aspartatedecarboxylase. For the purposes of the invention, it is not requiredthat the reactions shown as being reversible proceed in the reversedirection.

FIG. 8 is a flow chart that shows an example of yet another process forproducing R,R monatin in accordance with the present invention. In thisexample, the process includes recycling the amino acid byproduct of theL-tryptophan reaction with the amino acid reactant of the R-MP reactionthrough a series of conversion reactions. In the specific examplediagrammed in FIG. 8, L-tryptophan is reacted reversibly with anL-aminotransferase and with alpha-ketoglutarate (α-KG) to produceindole-3-pyruvate and L-glutamate. Indole-3-pyruvate is reversiblyreacted with pyruvate and an R-specific aldolase and converted toR-alpha-keto acid monatin (R-MP). R-MP is reacted in a reversible mannerwith a D-aminotransferase and D-alanine to form R,R monatin andpyruvate. An L-alanine aminotransferase and pyruvate are used toreversibly convert the L-glutamate that was a product of theL-aminotransferase reaction back to α-KG, with L-alanine as aco-product. An alanine racemase reversibly converts the L-alanine to theD-alanine that is useful in the third reaction, (the D-aminotransferasereaction. For the purposes of the invention, it is not required that thereactions shown as being reversible proceed in the reverse direction.

FIG. 9 is a block diagram of a computer system.

FIG. 10 is a flow diagram illustrating one embodiment of a process forcomparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database.

FIG. 11 is a flow diagram illustrating one embodiment of a process in acomputer for determining whether two sequences are homologous.

FIG. 12 is a flow diagram illustrating one embodiment of an identifierprocess 300 for detecting the presence of a feature in a sequence.

FIGS. 13 and 14 together illustrate the activities of 58 differentaldolases (each identified by its specific SEQ ID number) in theformation of monatin precursor (MP) as measured by LC/MS/MS.

FIG. 15 illustrates the effect of dithiothreitol on the production ofmonatin by the polypeptide with aldolase activity of SEQ ID NO:88.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

A number of embodiments have been described above and are described inmore detail infra. Embodiments of the invention include one or more ofthe described aspects.

ABBREVIATIONS AND TERMS

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. As used herein,“including” means “comprising.” In addition, the singular forms “a” or“an” or “the” include plural references unless the context clearlydictates otherwise. For example, reference to “comprising a protein”includes one or a plurality of such proteins, and reference to“comprising the cell” includes reference to one or more cells andequivalents thereof known to those skilled in the art, and so forth. Theterm “about” encompasses the range of experimental error that occurs inany measurement. Unless otherwise stated, all measurement numbers arepresumed to have the word “about” in front of them even if the word“about” is not expressly used.

Conservative substitution: a substitution of one amino acid for anotheramino acid in a polypeptide, which substitution has little to no impacton the activity of the polypeptide. The substitution is consideredconservative independent of whether the exchanged amino acids appearstructurally or functionally similar. For example, ideally, a tryptophanaminotransferase polypeptide including one or more conservativesubstitutions retains tryptophan aminotransferase activity. Apolypeptide can be produced to contain one or more conservativesubstitutions by manipulating the nucleotide sequence that encodes thatpolypeptide using, for example, standard procedures such assite-directed mutagenesis or PCR or other methods known to those in theart.

Non-limiting examples of amino acids which may be substituted for anoriginal amino acid in a protein and which may be regarded asconservative substitutions if there is little to no impact on theactivity of the polypeptide include: Ala substituted with ser or thr;arg substituted with gln, his, or lys; asn substituted with glu, gln,lys, his, asp; asp substituted with asn, glu, or gln; cys substitutedwith ser or ala; gln substituted with asn, glu, lys, his, asp, or arg;glu substituted with asn, gln lys, or asp; gly substituted with pro; hissubstituted with asn, lys, gln, arg, tyr; ile substituted with leu, met,val, phe; leu substituted with ile, met, val, phe; lys substituted withasn, glu, gln, his, arg; met substituted with ile, leu, val, phe; phesubstituted with trp, tyr, met, ile, or leu; ser substituted with thr,ala; thr substituted with ser or ala; trp substituted with phe, tyr; tyrsubstituted with his, phe, or trp; and val substituted with met, ile,leu.

Further information about conservative substitutions can be found in,among other locations, Ben-Bassat et al., (J. Bacteriol. 169:751-7,1987), O'Regan et al., (Gene 77:237-51, 1989), Sahin-Toth et al.,(Protein Sci. 3:240-7, 1994), Hochuli et al., (Bio/Technology 6:1321-5,1988), WO 00/67796 (Curd et al.) and in standard textbooks of geneticsand molecular biology.

Derived: For purposes of the specification and claims, a substance is“derived” from an organism or source if any one or more of the followingare true: 1) the substance is present in the organism/source; 2) thesubstance is removed from the native host; or, 3) the substance isremoved from the native host and is evolved, for example, bymutagenesis.

Isolated: The term “isolated” as used herein refers to any substanceremoved from its native host; the substance need not be purified. Forexample “isolated nucleic acid” refers to a naturally-occurring nucleicacid that is not immediately contiguous with both of the sequences withwhich it is immediately contiguous (one on the 5′ end and one on the 3′end) in the naturally-occurring genome of the organism from which it isderived. For example, an isolated nucleic acid can be, withoutlimitation, a recombinant DNA molecule of any length, provided one ofthe nucleic acid sequences normally found immediately flanking thatrecombinant DNA molecule in a naturally-occurring genome is removed orabsent. Thus, an isolated nucleic acid includes, without limitation, arecombinant DNA that exists as a separate molecule (such as a cDNA or agenomic DNA fragment produced by PCR or restriction endonucleasetreatment) independent of other sequences as well as recombinant DNAthat is incorporated into a vector, an autonomously replicating plasmid,a virus (such as a retrovirus, adenovirus, or herpes virus), or into thegenomic DNA of a prokaryote or eukaryote. In addition, an isolatednucleic acid can include a recombinant DNA molecule that is part of ahybrid or fusion nucleic acid sequence.

As used herein, the term “isolated” means that the material (such as aprotein or nucleic acid in accordance with the invention) is removedfrom its original environment (such as the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotideor polypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition and still be isolated inthat such vector or composition is not part of its natural environment.

The term “isolated” as used herein with reference to nucleic acid alsoincludes any non-naturally-occurring nucleic acid becausenon-naturally-occurring nucleic acid sequences are not found in natureand do not have immediately contiguous sequences in anaturally-occurring genome. For example, non-naturally-occurring nucleicacid such as an engineered nucleic acid is considered to be isolatednucleic acid. Engineered nucleic acid can be made using common molecularcloning or chemical nucleic acid synthesis techniques. Isolatednon-naturally-occurring nucleic acid can be independent of othersequences, or incorporated into a vector, an autonomously replicatingplasmid, a virus (such as a retrovirus, adenovirus, or herpes virus), orthe genomic DNA of a prokaryote or eukaryote. In addition, anon-naturally-occurring nucleic acid can include a nucleic acid moleculethat is part of a hybrid or fusion nucleic acid sequence.

Purified: The term “purified” as used herein does not require absolutepurity, but rather is intended as a relative term. Thus, for example, apurified polypeptide or nucleic acid preparation can be one in which thesubject polypeptide or nucleic acid is at a higher concentration thanthe polypeptide or nucleic acid would be in its natural environmentwithin an organism or at a higher concentration than in the environmentfrom which it was removed.

Individual nucleic acids obtained from a library have beenconventionally purified to electrophoretic homogeneity. The sequencesobtained from these clones could not be obtained directly either fromthe library or from total human DNA. The purified nucleic acids inaccordance with the invention have been purified from the remainder ofthe genomic DNA in the organism by at least 10⁴-10⁶ fold. In someembodiments, the term “purified” includes nucleic acids which have beenpurified from the remainder of the genomic DNA or from other sequencesin a library or other environment by at least one order of magnitude,such as, in some embodiments, two or three orders, or, four or fiveorders of magnitude.

Amino acid: “Amino acid” or “amino acid sequence” as used herein referto an oligopeptide, peptide, polypeptide, or protein sequence, or to afragment, portion, or subunit of any of these and to naturally occurringor synthetic molecules. “Amino acid” or “amino acid sequence” include anoligopeptide, peptide, polypeptide, or protein sequence, or to afragment, portion, or subunit of any of these, and to naturallyoccurring or synthetic molecules. The term “polypeptide” as used herein,refers to amino acids joined to each other by peptide bonds or modifiedpeptide bonds, i.e., peptide isosteres and may contain modified aminoacids other than the 20 gene-encoded amino acids. The polypeptides maybe modified by either natural processes, such as post-translationalprocessing, or by chemical modification techniques which are well knownin the art. Modifications can occur anywhere in the polypeptide,including the peptide backbone, the amino acid side-chains and the aminoor carboxyl termini. It will be appreciated that the same type ofmodification may be present in the same or varying degrees at severalsites in a given polypeptide. Also, a given polypeptide may have manytypes of modifications. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of a phosphatidylinositol, cross-linkingcyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristolyation, oxidation,pegylation, glucan hydrolase processing, phosphorylation, prenylation,racemization, selenoylation, sulfation and transfer-RNA mediatedaddition of amino acids to protein such as arginylation. (See Creighton,T. E., Proteins—Structure and Molecular Properties 2nd Ed., W.H. Freemanand Company, New York (1993); Posttranslational Covalent Modification ofProteins, B. C. Johnson, Ed., Academic Press, New York, pp. 1-12(1983)). The peptides and polypeptides in accordance with the inventionalso include all “mimetic” and “peptidomimetic” forms, as described infurther detail, below.

Polypeptide Having an Aldolase Activity: By a “polypeptide having analdolase activity” is meant a polypeptide that either by itself, or inassociation with one or more additional polypeptides (having the same ora different sequence), is a protein with the enzymatic activity of analdolase.

Recombinant: “Recombinant” polypeptides or proteins refer topolypeptides or proteins produced by recombinant DNA techniques; i.e.,produced from cells transformed by an exogenous DNA construct encodingthe desired polypeptide or protein. “Synthetic” polypeptides or proteinare those prepared by chemical synthesis. Solid-phase chemical peptidesynthesis methods can also be used to synthesize the polypeptide orfragments in accordance with the invention. Such method have been knownin the art since the early 1960's (Merrifield, R. B., J. Am. Chem. Soc.,85:2149-2154, 1963) (See also Stewart, J. M. and Young, J. D., SolidPhase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill.,pp. 11-12)) and have recently been employed in commercially availablelaboratory peptide design and synthesis kits (Cambridge ResearchBiochemicals). Such commercially available laboratory kits havegenerally utilized the teachings of H. M. Geysen et al, Proc. Natl.Acad. Sci., USA, 81:3998 (1984) and provide for synthesizing peptidesupon the tips of a multitude of “rods” or “pins” all of which areconnected to a single plate.

Substantially identical: The phrase “substantially identical” in thecontext of two nucleic acids or polypeptides, refers to two or moresequences that have, such as at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more nucleotide or amino acid residue (sequence)identity, when compared and aligned for maximum correspondence, asmeasured using one of the known sequence comparison algorithms or byvisual inspection. In other embodiments, the substantial identity existsover a region of at least about 100 or more residues and most commonlythe sequences are substantially identical over at least about 150 to 200or more residues. In some embodiments, the sequences are substantiallyidentical over the entire length of the coding regions.

Additionally a “substantially identical” amino acid sequence is asequence that differs from a reference sequence by one or moreconservative or non-conservative amino acid substitutions, deletions, orinsertions. In some embodiments, the substitution occurs at a site thatis not the active site of the molecule, or, alternatively thesubstitution occurs at a site that is the active site of the molecule,provided that the polypeptide essentially retains its functional(enzymatic) properties. A conservative amino acid substitution, forexample, substitutes one amino acid for another of the same class (suchas substitution of one hydrophobic amino acid, such as isoleucine,valine, leucine, or methionine, for another, or substitution of onepolar amino acid for another, such as substitution of arginine forlysine, glutamic acid for aspartic acid or glutamine for asparagine).One or more amino acids can be deleted, for example, from an aldolase,such as pyruvate aldolase, HMG and/or KHG aldolase polypeptide,resulting in modification of the structure of the polypeptide, withoutsignificantly altering its biological activity. For example, amino- orcarboxyl-terminal amino acids that are not required for aldolase, suchas pyruvate aldolase, such as HMG and/or KHG aldolase enzyme, biologicalactivity can be removed. Modified polypeptide sequences in accordancewith the invention can be assayed for aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzyme, biological activity byany number of methods, including contacting the modified polypeptidesequence with a substrate and determining whether the modifiedpolypeptide decreases the amount of specific substrate in the assay orincreases the bioproducts of the enzymatic reaction of a functionalaldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolasepolypeptide with the substrate.

Fragment: A “fragment” as used herein with regard to a protein orpolypeptide or nucleic acid is a portion of the protein, polypeptide ornucleic acid, respectively. Fragments can have the same or substantiallythe same amino acid or nucleic acid sequence as the longer protein,polypeptide or nucleic acid sequence from which the fragment is derived.Fragments which have different three dimensional structures as comparedto that of the longer protein, polypeptide or nucleic acid are alsoincluded. An example of this, is a “pro-form” molecule, such as a lowactivity proprotein that can be modified by cleavage to produce a matureenzyme with significantly higher activity. A fragment of a protein orpolypeptide can be an enzymatically active portion of a protein orpolypeptide.

Stereoinverting aminotransferase: A “stereoinverting aminotransferase”is a polypeptide capable of preferentially or selectively producing achiral amino acid product (such as monatin) while using an oppositechirality substrate as the amino donor. For example, a stereoinvertingaminotransferase may be a D-phenylglycine aminotransferase (also calledD-4-hydroxyphenylglycine aminotransferase) that preferentially orselectively uses L-glutamate as a substrate to produce R,R monatin.Non-limiting examples of stereoinverting aminotransferases includeD-methionine aminotransferase (EC 2.6.1.41) and enzymes havingD-phenylglycine aminotransferase activity or D-4-hydroxyphenylglycineaminotransferase activity.

The invention provides polypeptides with aldolase, including pyruvateactivity such as, without limitation, HMG and/or KHG aldolase activity,polynucleotides encoding them, and methods of making and using thesepolynucleotides and polypeptides. In some embodiments, the inventionalso provides aldolase enzymes, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzymes, polynucleotides encoding these enzymes, theuse of such polynucleotides and polypeptides.

In some embodiments, the invention provides modified or evolvedaldolases, such as pyruvate aldolases, HMG and/or KHG aldolases, with anincreased specific activity as compared to the unmodified or unevolvedaldolases, respectively.

In some embodiments, aldolases, such as a pyruvate aldolase, such as,without limitation a HMG and/or a KHG aldolase, are provided thatfacilitate the production of a 3,4-substituted 2-keto-glutarate. In oneembodiment, the invention provides a method of making a 3,4-substituted2-keto-glutarate comprising: (a) providing a polypeptide having analdolase activity, such as a pyruvate aldolase activity, such as,without limitation, a HMG aldolase and/or a KMG aldolase activity; (b)providing a donor and an acceptor compound; and (c) contacting thepolypeptide of step (a) with the compounds of step (b) under conditionswherein the aldolase catalyzes the synthesis of a 3,4-substituted2-keto-glutarate, wherein optionally the donor and the acceptor are apyruvate or a pyruvate donor and an α-keto acid acceptor, a ketoneand/or an aldehyde.

In another embodiment of the invention, a pyruvate aldolase, such as aHMG and/or a KHG aldolase, can be used in conjunction with aD-aminotransferase to make a 4-substituted D-glutamic acid or aderivative thereof. A 4-substituted D-glutamic acid and/or a derivativethereof can be used as an antibiotic, as these compounds have been foundto inhibit bacterial glutamate racemase. In one embodiment, theinvention provides a method of making a 4-substituted D-glutamic acidcomprising: (a) providing a polypeptide having an aldolase activity,such as a pyruvate aldolase activity, such as, without limitation, a HMGaldolase and/or a KMG aldolase activity; (b) providing an α-keto acidacceptor and a pyruvate or a pyruvate donor; and (c) contacting thepolypeptide of step (a) with the compounds of step (b) under conditionswherein the aldolase catalyzes the synthesis of a 4-substitutedD-glutamic acid, wherein optionally the polypeptide has pyruvatealdolase, HMG aldolase and/or KHG aldolase activity and whereinoptionally the method further comprises use of a D-aminotransferase.

In some embodiments the invention provides compositions (such as enzymepreparations, foods and food additives, feeds and feed additives,beverage and beverage additives, drugs and drug additives, and dietarysupplements) comprising the enzymes, polypeptides or polynucleotides inaccordance with the invention. These compositions can be formulated in avariety of forms, such as liquids, gels, pills, tablets, sprays, films,micelles, powders, food, feed pellets or encapsulated forms, includingnanoencapsulated forms.

Assays for measuring aldolase activity, including pyruvate activity suchas, without limitation, HMG and/or KHG aldolase activity, such as fordetermining if a polypeptide has aldolase activity, including pyruvateactivity such as, without limitation, HMG and/or KHG aldolase activity,are well known in the art and are within the scope in accordance withthe invention; see E. E. Dekker & R. P. Kitson, J. Biol. Chem. 267,10507-10514, 1992; Taha T S, Deits T L, Purification andcharacterization of 2-keto-3-deoxy-6-phosphogluconate aldolase fromAzotobacter vinelandii: evidence that the enzyme is bifunctional towards2-keto-4-hydroxy glutarate cleavage, Biochem Biophys Res Commun 1994Apr. 15; 200(1):459-66; Dekker E E, Kobes R D, Grady S R,2-keto-4-hydroxyglutarate aldolase from bovine liver, Methods Enzymol.1975; 42:280-5; Dekker E E, Nishihara H, Grady S R, Methods Enzymol.1975; 42:285-90, 2-keto-4-hydroxyglutarate aldolase from Escherichiacoli; Nishihara H, Dekker E E, Biochim Biophys Acta. 1969 Jul. 8;185(1):255-7, A stereospecific 2-keto-4-hydroxyglutarate aldolase fromEscherichia coli. One example of a suitable assay for determining if apolypeptide has aldolase activity, such as pyruvate aldolase, such asHMG and/or KHG aldolase activity is described in Example 3.

In some embodiments, the aldolases of the invention can be usedeffectively at a variety of pH conditions, including for example, from arange of about 3.0 to about 12.0. In other embodiments, the aldolases ofthe invention can be used at about pH is 3.0, 4.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, or about12.0. Reaction conditions conducted under acidic or alkaline conditionsalso can be advantageous, such as in some industrial or pharmaceuticalapplications of enzymes in accordance with the invention.

The invention provides aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase polypeptides in accordance with the invention in avariety of forms and formulations. In the methods in accordance with theinvention, aldolase, such as pyruvate aldolase, such as HMG and/or KHGaldolase polypeptides in accordance with the invention are used in avariety of forms and formulations. For example, purified aldolase, suchas pyruvate aldolase, such as HMG and/or KHG aldolase polypeptides canbe used in enzyme preparations deployed in the production of R-2-hydroxy2-(indol-3ylmethyl)-4-keto glutaric acid (R-MP) and certainstereoisomers of monatin, such as R,R and S,R monatin, and saltsthereof, as well as certain stereoisomers of monatin derivatives, suchas the R,R and S,R monatin derivative configurations, and salts thereof,or in pharmaceutical or dietary aid applications. Alternatively, theenzymes in accordance with the invention can be used directly inprocesses to produce R-2-hydroxy 2-(indol-3ylmethyl)-4-keto glutaricacid (R-MP) and certain stereoisomers of monatin, such as R,R and S,Rmonatin, and salts thereof, as well as certain stereoisomers of monatinderivatives, such as the R,R and S,R monatin derivative configurations,and salts thereof, to process foods, liquids or feeds, and the like.

In some embodiments, aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase polypeptides in accordance with the invention can beexpressed in a microorganism using procedures known in the art. In someembodiments, the aldolase, such as pyruvate aldolase, such as HMG and/orKHG aldolase polypeptides in accordance with the invention can beimmobilized on a solid support prior to use in the methods in accordancewith the invention. Methods for immobilizing enzymes on solid supportsare commonly known in the art, for example J. Mol. Cat. B: Enzymatic 6(1999) 29-39; Chivata et al. Biocatalysis: Immobilized cells andenzymes, J. Mol. Cat. 37 (1986) 1-24: Sharma et al., ImmobilizedBiomaterials Techniques and applications, Angew. Chem. Int. Ed. Engl. 21(1982) 837-54: Laskin (Ed.), Enzymes and Immobilized Cells inBiotechnology.

Nucleic Acids, Probes and Inhibitory Molecules

The invention provides isolated and recombinant nucleic acids, such assee Sequence Listing; nucleic acids encoding polypeptides, including thepolynucleotide sequences in accordance with the invention, such as seeSequence Listing; including expression cassettes such as expressionvectors and various cloning vehicles comprising nucleic acids inaccordance with the invention. In some embodiments, the invention alsoincludes methods for discovering, identifying or isolated new aldolase,such as pyruvate aldolase, HMG and/or KHG aldolase polypeptide sequencesusing the nucleic acids in accordance with the invention. In someembodiments, the invention also includes methods for inhibiting theexpression of aldolase, such as pyruvate aldolase, such as HMG and/orKHG aldolase encoding genes and transcripts using the nucleic acids inaccordance with the invention.

Also provided are methods for modifying the nucleic acids in accordancewith the invention, including making variants of nucleic acids inaccordance with the invention, by, such as synthetic ligationreassembly, optimized directed evolution system and/or saturationmutagenesis such as gene site saturation mutagenesis (GSSM). The term“saturation mutagenesis”, Gene Site Saturation Mutagenesis, or “GSSM”includes a method that uses degenerate oligonucleotide primers tointroduce point mutations into a polynucleotide, as described in detail,below. The term “optimized directed evolution system” or “optimizeddirected evolution” includes a method for reassembling fragments ofrelated nucleic acid sequences, such as related genes, and explained indetail, below. The term “synthetic ligation reassembly” or “SLR”includes a method of ligating oligonucleotide fragments in anon-stochastic fashion, and explained in detail, below. The term“variant” refers to polynucleotides or polypeptides in accordance withthe invention modified at one or more base pairs, codons, introns,exons, or amino acid residues (respectively) yet still retain thebiological activity of an aldolase, such as pyruvate aldolase, HMGand/or KHG aldolase in accordance with the invention. Variants can beproduced by any number of means included methods such as, for example,error-prone PCR, shuffling, oligonucleotide-directed mutagenesis,assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassettemutagenesis, recursive ensemble mutagenesis, exponential ensemblemutagenesis, site-specific mutagenesis, gene reassembly, GSSM and anycombination thereof.

The nucleic acids in accordance with the invention can be made, isolatedand/or manipulated by, such as cloning and expression of cDNA libraries,amplification of message or genomic DNA by PCR, and the like. Forexample, sequences in accordance with the invention were initiallyderived from environmental sources. Thus, in some embodiments, theinvention provides aldolase-, such as pyruvate aldolase-, such as HMGand/or KHG aldolase enzyme-encoding nucleic acids, and the polypeptidesencoded by them, preferably derived from a common source, such as anenvironmental, mixed culture, or a bacterial source.

In practicing the methods in accordance with the invention, homologousgenes can be modified by manipulating a template nucleic acid, asdescribed herein. In some embodiments, the invention can be practiced inconjunction with any method or protocol or device known in the art,which are well described in the scientific and patent literature.

The phrases “nucleic acid” or “nucleic acid sequence” as used hereinrefer to an oligonucleotide, nucleotide, polynucleotide, or to afragment of any of these, to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent asense or antisense (complementary) strand, to peptide nucleic acid(PNA), or to any DNA-like or RNA-like material, natural or synthetic inorigin. The phrases “nucleic acid” or “nucleic acid sequence” includesoligonucleotide, nucleotide, polynucleotide, or to a fragment of any ofthese, to DNA or RNA (such as mRNA, rRNA, tRNA, iRNA) of genomic orsynthetic origin which may be single-stranded or double-stranded and mayrepresent a sense or antisense strand, to peptide nucleic acid (PNA), orto any DNA-like or RNA-like material, natural or synthetic in origin,including, such as iRNA, ribonucleoproteins (such as double strandediRNAs, such as iRNPs). The term encompasses nucleic acids, i.e.,oligonucleotides, containing known analogues of natural nucleotides. Theterm also encompasses nucleic-acid-like structures with syntheticbackbones, see such as Mata (1997) Toxicol. Appl. Pharmacol.144:189-197; Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag(1996) Antisense Nucleic Acid Drug Dev 6:153-156. “Oligonucleotide”includes either a single stranded polydeoxynucleotide or twocomplementary polydeoxynucleotide strands which may be chemicallysynthesized. Such synthetic oligonucleotides have no 5′ phosphate andthus will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide can ligate to a fragment that has not beendephosphorylated.

A “coding sequence of” or a “nucleotide sequence encoding” a particularpolypeptide or protein, is a nucleic acid sequence which can betranscribed and translated into a polypeptide or protein when placedunder the control of appropriate regulatory sequences. The term “gene”means the segment of DNA involved in producing a polypeptide chain; itincludes regions preceding and following the coding region (leader andtrailer) as well as, where applicable, intervening sequences (introns)between individual coding segments (exons). A promoter sequence is“operably linked to” a coding sequence when RNA polymerase whichinitiates transcription at the promoter will transcribe the codingsequence into mRNA. “Operably linked” as used herein refers to afunctional relationship between two or more nucleic acid (such as DNA)segments. It can refer to the functional relationship of transcriptionalregulatory sequence to a transcribed sequence. For example, a promoteris operably linked to a coding sequence, such as a nucleic acid inaccordance with the invention, if it stimulates or modulates thetranscription of the coding sequence in an appropriate host cell orother expression system. Generally, promoter transcriptional regulatorysequences that are operably linked to a transcribed sequence arephysically contiguous to the transcribed sequence, i.e., they arecis-acting. However, some transcriptional regulatory sequences, such asenhancers, need not be physically contiguous or located in closeproximity to the coding sequences whose transcription they enhance.

The term “expression cassette” as used herein refers to a nucleotidesequence which is capable of affecting expression of a structural gene(i.e., a protein coding sequence, such as an aldolase, such as pyruvatealdolase, HMG and/or KHG aldolase enzyme in accordance with theinvention) in a host compatible with such sequences. Expressioncassettes include at least a promoter operably linked with thepolypeptide coding sequence; and, optionally, with other sequences, suchas transcription termination signals. Additional factors necessary orhelpful in effecting expression may also be used, such as enhancers,alpha-factors. Thus, expression cassettes also include plasmids,expression vectors, recombinant viruses, any form of recombinant “nakedDNA” vector, and the like. A “vector” comprises a nucleic acid which caninfect, transfect, transiently or permanently transduce a cell. It willbe recognized that a vector can be a naked nucleic acid, or a nucleicacid complexed with protein or lipid. The vector optionally comprisesviral or bacterial nucleic acids and/or proteins, and/or membranes (suchas a cell membrane, a viral lipid envelope, etc.). Vectors include, butare not limited to replicons (such as RNA replicons, bacteriophages) towhich fragments of DNA may be attached and become replicated. Vectorsthus include, but are not limited to RNA, autonomous self-replicatingcircular or linear DNA or RNA (such as plasmids, viruses, and the like,see U.S. Pat. No. 5,217,879), and include both the expression andnon-expression plasmids. Where a recombinant microorganism or cellculture is described as hosting an “expression vector” this includesboth extra-chromosomal circular and linear DNA and DNA that has beenincorporated into the host chromosome(s). Where a vector is beingmaintained by a host cell, the vector may either be stably replicated bythe cells during mitosis as an autonomous structure, or is incorporatedwithin the host's genome.

As used herein, the term “recombinant” encompasses nucleic acidsadjacent to a “backbone” nucleic acid to which it is not adjacent in itsnatural environment. In some embodiments, to be “enriched” the nucleicacids will represent about 5% or more of the number of nucleic acidinserts in a population of nucleic acid backbone molecules. Backbonemolecules according to the invention include nucleic acids such asexpression vectors, self-replicating nucleic acids, viruses, integratingnucleic acids and other vectors or nucleic acids used to maintain ormanipulate a nucleic acid insert of interest. In some embodiments, theenriched nucleic acids represent about 15% or more of the number ofnucleic acid inserts in the population of recombinant backbonemolecules. In some embodiments, the enriched nucleic acids representabout 50% or more of the number of nucleic acid inserts in thepopulation of recombinant backbone molecules. In some embodiments, theenriched nucleic acids represent about 90% or more of the number ofnucleic acid inserts in the population of recombinant backbonemolecules.

One embodiment of the invention is an isolated, synthetic or recombinantnucleic acid comprising one of the sequences in accordance with theinvention, or a fragment comprising at least 10, 15, 20, 25, 30, 35, 40,50, 75, 100, 150, 200, 300, 400, or 500 or more consecutive bases of anucleic acid in accordance with the invention. The isolated, syntheticor recombinant nucleic acids may comprise DNA, including cDNA, genomicDNA and synthetic DNA. The DNA may be double-stranded or single-strandedand if single stranded may be the coding strand or non-coding(anti-sense) strand. Alternatively, the isolated, synthetic orrecombinant nucleic acids comprise RNA.

The isolated, synthetic or recombinant nucleic acids in accordance withthe invention may be used to prepare one of the polypeptides inaccordance with the invention, or fragments comprising at least 5, 10,15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 or more consecutive aminoacids of one of the polypeptides in accordance with the invention.Accordingly, another embodiment of the invention is an isolated,synthetic or recombinant nucleic acid which encodes one of thepolypeptides in accordance with the invention, or fragments comprisingat least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 or moreconsecutive amino acids of one of the polypeptides in accordance withthe invention. The coding sequences of these nucleic acids may beidentical to one of the coding sequences of one of the nucleic acids inaccordance with the invention or may be different coding sequences whichencode one of the in accordance with the invention having at least 5,10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 or more consecutiveamino acids of one of the polypeptides in accordance with the invention,as a result of the redundancy or degeneracy of the genetic code. Thegenetic code is well known to those of skill in the art and can beobtained, such as on page 214 of B. Lewin, Genes VI, Oxford UniversityPress, 1997.

The isolated nucleic acid which encodes one of the polypeptides of theinvention and sequences substantially identical thereto, may include,but is not limited to: the coding sequence of a nucleic acid inaccordance with the invention and additional coding sequences, such asleader sequences or proprotein sequences and non-coding sequences, suchas introns or non-coding sequences 5′ and/or 3′ of the coding sequence.Thus, as used herein, the term “polynucleotide encoding a polypeptide”encompasses a polynucleotide which includes the coding sequence for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequence.

Alternatively, the nucleic acid sequences of the invention and sequencessubstantially identical thereto, may be mutagenized using conventionaltechniques, such as site directed mutagenesis, or other techniquesfamiliar to those skilled in the art, to introduce silent changes intothe polynucleotides o in accordance with the invention. As used herein,“silent changes” include, for example, changes which do not alter theamino acid sequence encoded by the polynucleotide. Such changes may bedesirable in order to increase the level of the polypeptide produced byhost cells containing a vector encoding the polypeptide by introducingcodons or codon pairs which occur frequently in the host organism.

The invention also relates to polynucleotides which have nucleotidechanges which result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptides in accordance with theinvention. Such nucleotide changes may be introduced using techniquessuch as site directed mutagenesis, random chemical mutagenesis,exonuclease III deletion and other recombinant DNA techniques.Alternatively, such nucleotide changes may be naturally occurringallelic variants which are isolated by identifying nucleic acids whichspecifically hybridize to probes comprising at least 10, 15, 20, 25, 30,35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutive bases of oneof the sequences in accordance with the invention (or the sequencescomplementary thereto) under conditions of high, moderate, or lowstringency as provided herein.

General Techniques

The nucleic acids used to practice this invention, whether RNA, siRNA,miRNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses orhybrids thereof, may be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly.Recombinant polypeptides (such as aldolase, such as pyruvate aldolase,such as HMG and/or KHG aldolase enzymes) generated from these nucleicacids can be individually isolated or cloned and tested for a desiredactivity. Any recombinant expression system can be used, includingbacterial, mammalian, yeast, insect or plant cell expression systems.

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, such as Adams(1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res.25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers(1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90;Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids, such as subcloning,labeling probes (such as random-primer labeling using Klenow polymerase,nick translation, amplification), sequencing, hybridization and the likeare well described in the scientific and patent literature, seeSambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols.1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS INMOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York(1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY:HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic AcidPreparation, Tijssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used topractice the methods in accordance with the invention is to clone fromgenomic samples, and, if desired, screen and re-clone inserts isolatedor amplified from, such as genomic clones or cDNA clones. Sources ofnucleic acid used in the methods in accordance with the inventioninclude genomic or cDNA libraries contained in, such as mammalianartificial chromosomes (MACS), see U.S. Pat. Nos. 5,721,118; 6,025,155;human artificial chromosomes, see Rosenfeld (1997) Nat. Genet.15:333-335; yeast artificial chromosomes (YAC); bacterial artificialchromosomes (BAC); P1 artificial chromosomes, see Woon (1998) Genomics50:306-316; P1-derived vectors (PACs), see Kern (1997) Biotechniques23:120-124; cosmids, recombinant viruses, phages or plasmids.

In some embodiments, a nucleic acid encoding a polypeptide in accordancewith the invention is assembled in appropriate phase with a leadersequence capable of directing secretion of the translated polypeptide orfragment thereof.

The invention provides fusion proteins and nucleic acids encoding them.A polypeptide in accordance with the invention can be fused to aheterologous peptide or polypeptide, such as N-terminal identificationpeptides which impart desired characteristics, such as increasedstability or simplified purification. Peptides and polypeptides inaccordance with the invention can also be synthesized and expressed asfusion proteins with one or more additional domains linked thereto for,such as producing a more immunogenic peptide, to more readily isolate arecombinantly synthesized peptide, to identify and isolate antibodiesand antibody-expressing B cells, and the like. Detection andpurification facilitating domains include, such as metal chelatingpeptides such as polyhistidine tracts and histidine-tryptophan modulesthat allow purification on immobilized metals, protein A domains thatallow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of a cleavable linker sequences suchas Factor Xa or enterokinase (Invitrogen, Carlsbad, Calif.) between apurification domain and the motif-comprising peptide or polypeptide tofacilitate purification. For example, an expression vector can includean epitope-encoding nucleic acid sequence linked to six histidineresidues followed by a thioredoxin and an enterokinase cleavage site(see such as Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998)Protein Expr. Purif. 12:404-414). The histidine residues facilitatedetection and purification while the enterokinase cleavage site providesmeans for purifying the epitope from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see such as Kroll (1993) DNA Cell. Biol., 12:441-53.

Transcriptional and Translational Control Sequences

The invention provides nucleic acid (such as DNA) sequences inaccordance with the invention operatively linked to expression (such astranscriptional or translational) control sequence(s), such as promotersor enhancers, to direct or modulate RNA synthesis/expression. Theexpression control sequence can be in an expression vector. Exemplarybacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL andtrp. Exemplary eukaryotic promoters include CMV immediate early, HSVthymidine kinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein I.

As used herein, the term “promoter” includes all sequences capable ofdriving transcription of a coding sequence in a cell, such as a plant oranimal cell. Thus, promoters used in the constructs in accordance withthe invention include cis-acting transcriptional control elements andregulatory sequences that are involved in regulating or modulating thetiming and/or rate of transcription of a gene. For example, a promotercan be a cis-acting transcriptional control element, including anenhancer, a promoter, a transcription terminator, an origin ofreplication, a chromosomal integration sequence, 5′ and 3′ untranslatedregions, or an intronic sequence, which are involved in transcriptionalregulation. These cis-acting sequences can interact with proteins orother biomolecules to carry out (turn on/off, regulate, modulate, etc.)transcription. “Constitutive” promoters are those that drive expressioncontinuously under most environmental conditions and states ofdevelopment or cell differentiation. “Inducible” or “regulatable”promoters direct expression of the nucleic acid in accordance with theinvention under the influence of environmental conditions ordevelopmental conditions. Examples of environmental conditions that mayaffect transcription by inducible promoters include anaerobicconditions, elevated temperature, drought, or the presence of light.

“Tissue-specific” promoters are transcriptional control elements thatare only active in particular cells or tissues or organs, such as inplants or animals. Tissue-specific regulation may be achieved by certainintrinsic factors which ensure that genes encoding proteins specific toa given tissue are expressed. Such factors are known to exist in mammalsand plants so as to allow for specific tissues to develop.

Promoters suitable for expressing a polypeptide in bacteria include theE. coli lac or trp promoters, the lad promoter, the lacZ promoter, theT3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter,the lambda PL promoter, promoters from operons encoding glycolyticenzymes such as 3-phosphoglycerate kinase (PGK), and the acidphosphatase promoter. Eukaryotic promoters include the CMV immediateearly promoter, the HSV thymidine kinase promoter, heat shock promoters,the early and late SV40 promoter, LTRs from retroviruses, and the mousemetallothionein-I promoter. Other promoters known to control expressionof genes in prokaryotic or eukaryotic cells or their viruses may also beused. Promoters suitable for expressing the polypeptide or fragmentthereof in bacteria include the E. coli lac or trp promoters, the lacIpromoter, the lacZ promoter, the T3 promoter, the T7 promoter, the gptpromoter, the lambda P_(R) promoter, the lambda P_(L) promoter,promoters from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK) and the acid phosphatase promoter.Fungal promoters include the α-factor promoter. Eukaryotic promotersinclude the CMV immediate early promoter, the HSV thymidine kinasepromoter, heat shock promoters, the early and late SV40 promoter, LTRsfrom retroviruses and the mouse metallothionein-I promoter. Otherpromoters known to control expression of genes in prokaryotic oreukaryotic cells or their viruses may also be used.

Tissue-Specific Plant Promoters

The invention provides expression cassettes that can be expressed in atissue-specific manner, such as that can express an aldolase, such aspyruvate aldolase, HMG and/or KHG aldolase enzyme in accordance with theinvention in a tissue-specific manner. In some embodiments, theinvention also provides plants or seeds that express an aldolase, suchas pyruvate aldolase, HMG and/or KHG aldolase enzyme in accordance withthe invention in a tissue-specific manner. The tissue-specificity can beseed specific, stem specific, leaf specific, root specific, fruitspecific and the like.

The term “plant” includes whole plants, plant parts (such as leaves,stems, flowers, roots, etc.), plant protoplasts, seeds and plant cellsand progeny of same. The class of plants which can be used in the methodin accordance with the invention is generally as broad as the class ofhigher plants amenable to transformation techniques, includingangiosperms (monocotyledonous and dicotyledonous plants), as well asgymnosperms. It includes plants of a variety of ploidy levels, includingpolyploid, diploid, haploid and hemizygous states. As used herein, theterm “transgenic plant” includes plants or plant cells into which aheterologous nucleic acid sequence has been inserted, such as thenucleic acids and various recombinant constructs (such as expressioncassettes) in accordance with the invention.

In some embodiments, a constitutive promoter such as the CaMV 35Spromoter can be used for expression in specific parts of the plant orseed or throughout the plant. For example, for overexpression, a plantpromoter fragment can be employed which will direct expression of anucleic acid in some or all tissues of a plant, such as a regeneratedplant. Such promoters are referred to herein as “constitutive” promotersand are active under most environmental conditions and states ofdevelopment or cell differentiation. Examples of constitutive promotersinclude the cauliflower mosaic virus (CaMV) 35S transcription initiationregion, the 1′- or 2′-promoter derived from T-DNA of Agrobacteriumtumefaciens, and other transcription initiation regions from variousplant genes known to those of skill. Such genes include, such as ACT11from Arabidopsis (Huang (1996) Plant Mol. Biol. 33:125-139); Cat3 fromArabidopsis (GenBank No. U43147, Zhong (1996) Mol. Gen. Genet.251:196-203); the gene encoding stearoyl-acyl carrier protein desaturasefrom Brassica napus (Genbank No. X74782, Solocombe (1994) Plant Physiol.104:1167-1176); GPc1 from maize (GenBank No. X15596; Martinez (1989) J.Mol. Biol. 208:551-565); the Gpc2 from maize (GenBank No. U45855,Manjunath (1997) Plant Mol. Biol. 33:97-112); plant promoters describedin U.S. Pat. Nos. 4,962,028; 5,633,440.

The invention uses tissue-specific or constitutive promoters derivedfrom viruses which can include, such as the tobamovirus subgenomicpromoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92:1679-1683; therice tungro bacilliform virus (RTBV), which replicates only in phloemcells in infected rice plants, with its promoter which drives strongphloem-specific reporter gene expression; the cassava vein mosaic virus(CVMV) promoter, with highest activity in vascular elements, in leafmesophyll cells, and in root tips (Verdaguer (1996) Plant Mol. Biol.31:1129-1139).

In some embodiments, the plant promoter directs expression of aldolase,such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme-expressing nucleic acid in a specific tissue, organ or cell type(i.e. tissue-specific promoters) or may be otherwise under more preciseenvironmental or developmental control or under the control of aninducible promoter. Examples of environmental conditions that may affecttranscription include anaerobic conditions, elevated temperature, thepresence of light, or sprayed with chemicals/hormones. For example, theinvention incorporates the drought-inducible promoter of maize (Busk(1997) supra); the cold, drought, and high salt inducible promoter frompotato (Kirch (1997) Plant Mol. Biol. 33:897 909).

In some embodiments, tissue-specific promoters promote transcriptiononly within a certain time frame of developmental stage within thattissue. See Blazquez (1998) Plant Cell 10:791-800, characterizing theArabidopsis LEAFY gene promoter. See also Cardon (1997) Plant J12:367-77, describing the transcription factor SPL3, which recognizes aconserved sequence motif in the promoter region of the A. thalianafloral meristem identity gene AP1; and Mandel (1995) Plant MolecularBiology, Vol. 29, pp 995-1004, describing the meristem promoter eIF4.Tissue specific promoters which are active throughout the life cycle ofa particular tissue can be used. In some embodiments, the nucleic acidsin accordance with the invention are operably linked to a promoteractive primarily only in cotton fiber cells. In some embodiments, thenucleic acids in accordance with the invention are operably linked to apromoter active primarily during the stages of cotton fiber cellelongation, such as described by Rinehart (1996) supra. The nucleicacids can be operably linked to the Fb12A gene promoter to bepreferentially expressed in cotton fiber cells (Ibid). See also, John(1997) Proc. Natl. Acad. Sci. USA 89:5769-5773; John, et al., U.S. Pat.Nos. 5,608,148 and 5,602,321, describing cotton fiber-specific promotersand methods for the construction of transgenic cotton plants.Root-specific promoters may also be used to express the nucleic acids inaccordance with the invention. Examples of root-specific promotersinclude the promoter from the alcohol dehydrogenase gene (DeLisle (1990)Int. Rev. Cytol. 123:39-60). Other promoters that can be used to expressthe nucleic acids in accordance with the invention include, such asovule-specific, embryo-specific, endosperm-specific,integument-specific, seed coat-specific promoters, or some combinationthereof; a leaf-specific promoter (see Busk (1997) Plant J. 11:12851295, describing a leaf-specific promoter in maize); the ORF13 promoterfrom Agrobacterium rhizogenes (which exhibits high activity in roots,see Hansen (1997) supra); a maize pollen specific promoter (see Guerrero(1990) Mol. Gen. Genet. 224:161 168); a tomato promoter active duringfruit ripening, senescence and abscission of leaves and, to a lesserextent, of flowers can be used (see Blume (1997) Plant J. 12:731 746); apistil-specific promoter from the potato SK2 gene (see Ficker (1997)Plant Mol. Biol. 35:425 431); the Blec4 gene from pea, which is activein epidermal tissue of vegetative and floral shoot apices of transgenicalfalfa making it a useful tool to target the expression of foreigngenes to the epidermal layer of actively growing shoots or fibers; theovule-specific BEL1 gene (see Reiser (1995) Cell 83:735-742, GenBank No.U39944); and/or, the promoter in Klee, U.S. Pat. No. 5,589,583,describing a plant promoter region is capable of conferring high levelsof transcription in meristematic tissue and/or rapidly dividing cells.

In some embodiments, plant promoters which are inducible upon exposureto plant hormones, such as auxins, are used to express the nucleic acidsin accordance with the invention. For example, the invention can use theauxin-response elements E1 promoter fragment (AuxREs) in the soybean(Glycine max L.) (Liu (1997) Plant Physiol. 115:397-407); theauxin-responsive Arabidopsis GST6 promoter (also responsive to salicylicacid and hydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); theauxin-inducible parC promoter from tobacco (Sakai (1996) Plant CellPhysiol. 37:906-913); a plant biotin response element (Streit (1997)Mol. Plant Microbe Interact. 10:933-937); and, the promoter responsiveto the stress hormone abscisic acid (Sheen (1996) Science274:1900-1902).

The nucleic acids in accordance with the invention can also be operablylinked to plant promoters which are inducible upon exposure to chemicalsreagents which can be applied to the plant, such as herbicides orantibiotics. For example, the maize In2-2 promoter, activated bybenzenesulfonamide herbicide safeners, can be used (De Veylder (1997)Plant Cell Physiol. 38:568-577); application of different herbicidesafeners induces distinct gene expression patterns, including expressionin the root, hydathodes, and the shoot apical meristem. Coding sequencecan be under the control of, such as a tetracycline-inducible promoter,such as described with transgenic tobacco plants containing the Avenasativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J.11:465-473); or, a salicylic acid-responsive element (Stange (1997)Plant J. 11:1315-1324). Using chemically—(such as hormone- orpesticide-) induced promoters, i.e., promoter responsive to a chemicalwhich can be applied to the transgenic plant in the field, expression ofa polypeptide in accordance with the invention can be induced at aparticular stage of development of the plant. Thus, the invention alsoprovides for transgenic plants containing an inducible gene encoding forpolypeptides in accordance with the invention whose host range islimited to target plant species, such as corn, rice, barley, soybean,tomato, wheat, potato or other crops, inducible at any stage ofdevelopment of the crop.

One of skill will recognize that a tissue-specific plant promoter maydrive expression of operably linked sequences in tissues other than thetarget tissue. Thus, In some embodiments, a tissue-specific promoter isone that drives expression preferentially in the target tissue or celltype, but may also lead to some expression in other tissues as well.

The nucleic acids in accordance with the invention can also be operablylinked to plant promoters which are inducible upon exposure to chemicalsreagents. These reagents include, such as herbicides, synthetic auxins,or antibiotics which can be applied, such as sprayed, onto transgenicplants. Inducible expression of the aldolase, such as pyruvate aldolase,such as HMG and/or KHG aldolase enzyme-producing nucleic acids inaccordance with the invention will allow the grower to select plantswith the optimal aldolase, such as pyruvate aldolase, such as HMG and/orKHG aldolase enzyme expression and/or activity. The development of plantparts can thus controlled. In this way the invention provides the meansto facilitate the harvesting of plants and plant parts. For example, invarious embodiments, the maize In2-2 promoter, activated bybenzenesulfonamide herbicide safeners, is used (De Veylder (1997) PlantCell Physiol. 38:568-577); application of different herbicide safenersinduces distinct gene expression patterns, including expression in theroot, hydathodes, and the shoot apical meristem. Coding sequences inaccordance with the invention are also under the control of atetracycline-inducible promoter, such as described with transgenictobacco plants containing the Avena sativa L. (oat) argininedecarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylicacid-responsive element (Stange (1997) Plant J. 11:1315-1324).

In some embodiments, proper polypeptide expression may requirepolyadenylation region at the 3′-end of the coding region. Thepolyadenylation region can be derived from the natural gene, from avariety of other plant (or animal or other) genes, or from genes in theAgrobacterial T-DNA.

Expression Vectors and Cloning Vehicles

The invention provides expression vectors and cloning vehiclescomprising nucleic acids in accordance with the invention, such assequences encoding the aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzymes in accordance with the invention. Expressionvectors and cloning vehicles in accordance with the invention cancomprise viral particles, baculovirus, phage, plasmids, phagemids,cosmids, fosmids, bacterial artificial chromosomes, viral DNA (such asvaccinia, adenovirus, foul pox virus, pseudorabies and derivatives ofSV40), P1-based artificial chromosomes, yeast plasmids, yeast artificialchromosomes, and any other vectors specific for specific hosts ofinterest (such as bacillus, Aspergillus and yeast). Vectors inaccordance with the invention can include chromosomal, non-chromosomaland synthetic DNA sequences. Large numbers of suitable vectors are knownto those of skill in the art, and are commercially available. Exemplaryvectors include: bacterial: pQE™ vectors (Qiagen, Valencia, Calif.),pBLUESCRIPT™ plasmids, pNH vectors, lambda-ZAP vectors (Stratagene, LaJolla, Calif.); ptrc99a, pKK223-3, pDR540, pRIT2T (GE Healthcare,Piscataway, N.J.), pET vectors (Novagen, Madison, Wis.); Eukaryotic:pXT1, pSG5 (Stratagene, La Jolla, Calif.), pSVK3, pBPV, pMSG, pSVLSV40(Pharmacia). However, any other plasmid or other vector may be used solong as they are replicable and viable in the host. Low copy number orhigh copy number vectors may be employed with the present invention.“Plasmids” can be commercially available, publicly available on anunrestricted basis, or can be constructed from available plasmids inaccord with published procedures. Equivalent plasmids to those describedherein are known in the art and will be apparent to the ordinarilyskilled artisan.

The expression vector can comprise a promoter, a ribosome binding sitefor translation initiation and a transcription terminator. The vectormay also include appropriate sequences for amplifying expression.Mammalian expression vectors can comprise an origin of replication, anynecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, transcriptional termination sequences, and 5′flanking non-transcribed sequences. In some embodiments, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements.

In some embodiments, the expression vectors contain one or moreselectable marker genes to permit selection of host cells containing thevector. Such selectable markers include genes encoding dihydrofolatereductase or genes conferring neomycin resistance for eukaryotic cellculture, genes conferring tetracycline or ampicillin resistance in E.coli, and the S. cerevisiae TRP1 gene. Promoter regions can be selectedfrom any desired gene using chloramphenicol transferase (CAT) vectors orother vectors with selectable markers.

In some embodiments, vectors for expressing the polypeptide or fragmentthereof in eukaryotic cells contain enhancers to increase expressionlevels. Enhancers are cis-acting elements of DNA that can be from about10 to about 300 bp in length. They can act on a promoter to increase itstranscription. Exemplary enhancers include the SV40 enhancer on the lateside of the replication origin by 100 to 270, the cytomegalovirus earlypromoter enhancer, the polyoma enhancer on the late side of thereplication origin, and the adenovirus enhancers.

A nucleic acid sequence can be inserted into a vector by a variety ofprocedures. In general, the sequence is ligated to the desired positionin the vector following digestion of the insert and the vector withappropriate restriction endonucleases. Alternatively, blunt ends in boththe insert and the vector may be ligated. A variety of cloningtechniques are known in the art, such as described in Ausubel et al.Current Protocols in Molecular Biology, John Wiley & Sons, Inc. 1997 andSambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed., ColdSpring Harbor Laboratory Press (1989). Such procedures and others aredeemed to be within the scope of those skilled in the art.

The vector can be in the form of a plasmid, a viral particle, or aphage. Other vectors include chromosomal, non-chromosomal and syntheticDNA sequences, derivatives of SV40; bacterial plasmids, phage DNA,baculovirus, yeast plasmids, vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies. A variety of cloning and expression vectors foruse with prokaryotic and eukaryotic hosts are described by, such asSambrook.

Particular bacterial vectors which can be used include the commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden), GEM1 (Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9(Qiagen, Valencia, Calif.), pD10, psiX174 pBLUESCRIPT II KS, pNH8A,pNH16a, pNH18A, pNH46A (Stratagene, La Jolla, Calif.), ptrc99a,pKK223-3, pKK233-3, DR540, pRIT5 (Pharmacia), pKK232-8, pET (Novagen,Madison, Wis.), and pCM7. Particular eukaryotic vectors include pSV2CAT,pOG44, pXT1, pSG (Stratagene, La Jolla, Calif.) pSVK3, pBPV, pMSG, andpSVL (Pharmacia). However, any other vector may be used as long as it isreplicable and viable in the host cell.

The nucleic acids in accordance with the invention can be expressed inexpression cassettes, vectors or viruses and transiently or stablyexpressed in plant cells and seeds. One exemplary transient expressionsystem uses episomal expression systems, such as cauliflower mosaicvirus (CaMV) viral RNA generated in the nucleus by transcription of anepisomal mini-chromosome containing supercoiled DNA, see Covey (1990)Proc. Natl. Acad. Sci. USA 87:1633-1637. Alternatively, codingsequences, i.e., all or sub-fragments of sequences in accordance withthe invention can be inserted into a plant host cell genome becoming anintegral part of the host chromosomal DNA. Sense or antisensetranscripts can be expressed in this manner. A vector comprising thesequences (such as promoters or coding regions) from nucleic acids inaccordance with the invention can comprise a marker gene that confers aselectable phenotype on a plant cell or a seed. For example, the markermay encode biocide resistance, such as antibiotic resistance, such asresistance to kanamycin, G418, bleomycin, hygromycin, or herbicideresistance, such as resistance to chlorosulfuron or Basta.

Expression vectors capable of expressing nucleic acids and proteins inplants are well known in the art, and can include, such as vectors fromAgrobacterium spp., potato virus X (see Angell (1997) EMBO J.16:3675-3684), tobacco mosaic virus (see Casper (1996) Gene 173:69-73),tomato bushy stunt virus (see Hillman (1989) Virology 169:42-50),tobacco etch virus (see Dolja (1997) Virology 234:243-252), bean goldenmosaic virus (see Morinaga (1993) Microbiol Immunol. 37:471-476),cauliflower mosaic virus (see Cecchini (1997) Mol. Plant MicrobeInteract. 10:1094-1101), maize Ac/Ds transposable element (see Rubin(1997) Mol. Cell. Biol. 17:6294-6302; Kunze (1996) Curr. Top. Microbiol.Immunol. 204:161-194), and the maize suppressor-mutator (Spm)transposable element (see Schlappi (1996) Plant Mol. Biol. 32:717-725);and derivatives thereof.

In some embodiments, the expression vector can have two replicationsystems to allow it to be maintained in two organisms, for example inmammalian or insect cells for expression and in a prokaryotic host forcloning and amplification. Furthermore, for integrating expressionvectors, the expression vector can contain at least one sequencehomologous to the host cell genome. It can contain two homologoussequences which flank the expression construct. The integrating vectorcan be directed to a specific locus in the host cell by selecting theappropriate homologous sequence for inclusion in the vector. Constructsfor integrating vectors are well known in the art.

Expression vectors in accordance with the invention may also include aselectable marker gene to allow for the selection of bacterial strainsthat have been transformed, such as genes which render the bacteriaresistant to drugs such as ampicillin, chloramphenicol, erythromycin,kanamycin, neomycin and tetracycline. Selectable markers can alsoinclude biosynthetic genes, such as those in the histidine, tryptophanand leucine biosynthetic pathways.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct RNAsynthesis. Particular named bacterial promoters include lacI, lacZ, T3,T7, gpt, lambda P_(R), P_(L), and trp. Eukaryotic promoters include CMVimmediate early, HSV thymidine kinase, early and late SV40, LTRs fromretrovirus and mouse metallothionein-I. Selection of the appropriatevector and promoter is well within the level of ordinary skill in theart. The expression vector also contains a ribosome binding site fortranslation initiation and a transcription terminator. The vector mayalso include appropriate sequences for amplifying expression. Promoterregions can be selected from any desired gene using chloramphenicoltransferase (CAT) vectors or other vectors with selectable markers. Inaddition, the expression vectors in some embodiments contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

Mammalian expression vectors may also comprise an origin of replication,any necessary ribosome binding sites, a polyadenylation site, splicedonor and acceptor sites, transcriptional termination sequences and 5′flanking nontranscribed sequences. In some embodiments, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required nontranscribed genetic elements.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells may also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 bp in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin by 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin and theadenovirus enhancers.

In addition, the expression vectors can contain one or more selectablemarker genes to permit selection of host cells containing the vector.Such selectable markers include genes encoding dihydrofolate reductaseor genes conferring neomycin resistance for eukaryotic cell culture,genes conferring tetracycline or ampicillin resistance in E. coli andthe S. cerevisiae TRP1 gene.

In some embodiments, the nucleic acid encoding one of the polypeptidesin accordance with the invention, and sequences substantially identicalthereto, or fragments comprising at least about 5, 10, 15, 20, 25, 30,35, 40, 50, 75, 100, or 150 or more consecutive amino acids thereof isassembled in appropriate phase with a leader sequence capable ofdirecting secretion of the translated polypeptide or fragment thereof.In some embodiments, the nucleic acid can encode a fusion polypeptide inwhich one of the polypeptides in accordance with the invention, orfragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, or 150 or more consecutive amino acids thereof is fused toheterologous peptides or polypeptides, such as N-terminal identificationpeptides which impart desired characteristics, such as increasedstability or simplified purification.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is ligated to thedesired position in the vector following digestion of the insert and thevector with appropriate restriction endonucleases. Alternatively, bluntends in both the insert and the vector may be ligated. A variety ofcloning techniques are disclosed in Ausubel et al. Current Protocols inMolecular Biology, John Wiley & Sons, Inc. 1997 and Sambrook et al.,Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring HarborLaboratory Press (1989). Such procedures and others are deemed to bewithin the scope of those skilled in the art.

The vector may be, for example, in the form of a plasmid, a viralparticle, or a phage. Other vectors include chromosomal, nonchromosomaland synthetic DNA sequences, derivatives of SV40; bacterial plasmids,phage DNA, baculovirus, yeast plasmids, vectors derived fromcombinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus and pseudorabies. A variety of cloning andexpression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor, N.Y., (1989).

Host Cells and Transformed Cells

The invention also provides transformed cells comprising nucleic acidsequences in accordance with the invention, such as sequences encodingaldolases, such as pyruvate aldolases, HMG and/or KHG aldolase enzymesin accordance with the invention, or vectors in accordance with theinvention. The host cell may be any of the host cells familiar to thoseskilled in the art, including prokaryotic cells, eukaryotic cells, suchas bacterial cells, fungal cells, yeast cells, mammalian cells, insectcells, or plant cells. Exemplary bacterial cells include any species ofStreptomyces, Staphylococcus, Pseudomonas or Bacillus, including E.coli, Bacillus subtilis, Pseudomonas fluorescens, Bacillus cereus, orSalmonella typhimurium. Exemplary fungal cells include any species ofAspergillus. Exemplary yeast cells include any species of Pichia,Saccharomyces, Schizosaccharomyces, or Schwanniomyces, including Pichiapastoris, Saccharomyces cerevisiae, or Schizosaccharomyces pombe.Exemplary insect cells include any species of Spodoptera or Drosophila,including Drosophila S2 and Spodoptera Sf9. Exemplary animal cellsinclude CHO, COS or Bowes melanoma or any mouse or human cell line. Theselection of an appropriate host is within the abilities of thoseskilled in the art. Techniques for transforming a wide variety of higherplant species are well known and described in the technical andscientific literature. See Weising (1988) Ann. Rev. Genet. 22:421-477;U.S. Pat. No. 5,750,870.

The vector can be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

In some embodiments, the nucleic acids or vectors in accordance with theinvention are introduced into the cells for screening, thus, the nucleicacids enter the cells in a manner suitable for subsequent expression ofthe nucleic acid. The method of introduction is largely dictated by thetargeted cell type. Exemplary methods include CaPO₄ precipitation,liposome fusion, lipofection (such as LIPOFECTIN™), electroporation,viral infection, etc. The candidate nucleic acids may stably integrateinto the genome of the host cell (for example, with retroviralintroduction) or may exist either transiently or stably in the cytoplasm(i.e. through the use of traditional plasmids, utilizing standardregulatory sequences, selection markers, etc.). As many pharmaceuticallyimportant screens require human or model mammalian cell targets,retroviral vectors capable of transfecting such targets can be used.

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes in accordancewith the invention. Following transformation of a suitable host strainand growth of the host strain to an appropriate cell density, theselected promoter may be induced by appropriate means (such astemperature shift or chemical induction) and the cells may be culturedfor an additional period to allow them to produce the desiredpolypeptide or fragment thereof.

Cells can be harvested by centrifugation, disrupted by physical orchemical means, and the resulting crude extract is retained for furtherpurification. Microbial cells employed for expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents. Suchmethods are well known to those skilled in the art. The expressedpolypeptide or fragment thereof can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the polypeptide. If desired,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides in accordance withthe invention may or may not also include an initial methionine aminoacid residue.

Cell-free translation systems can also be employed to produce apolypeptide in accordance with the invention. Cell-free translationsystems can use mRNAs transcribed from a DNA construct comprising apromoter operably linked to a nucleic acid encoding the polypeptide orfragment thereof. In some embodiments, the DNA construct may belinearized prior to conducting an in vitro transcription reaction. Thetranscribed mRNA is then incubated with an appropriate cell-freetranslation extract, such as a rabbit reticulocyte extract, to producethe desired polypeptide or fragment thereof.

The expression vectors can contain one or more selectable marker genesto provide a phenotypic trait for selection of transformed host cellssuch as dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, or such as tetracycline or ampicillin resistance in E.coli.

Host cells containing the polynucleotides of interest, such as nucleicacids in accordance with the invention, can be cultured in conventionalnutrient media modified as appropriate for activating promoters,selecting transformants or amplifying genes. The culture conditions,such as temperature, pH and the like, are those previously used with thehost cell selected for expression and will be apparent to the ordinarilyskilled artisan. The clones which are identified as having the specifiedenzyme activity may then be sequenced to identify the polynucleotidesequence encoding an enzyme having the enhanced activity.

The invention provides methods for overexpressing recombinant aldolases,such as pyruvate aldolases, such as HMG and/or KHG aldolase enzymes incells comprising expressing a vector comprising a nucleic acid inaccordance with the invention, such as a nucleic acid comprising anucleic acid sequence with at least about 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to a sequence in accordance with theinvention over a region of at least about 100 residues, wherein thesequence identities are determined by analysis with a sequencecomparison algorithm or by visual inspection, or, a nucleic acid thathybridizes under stringent conditions to a nucleic acid sequence inaccordance with the invention, or a subsequence thereof. Theoverexpression can be effected by any means, such as use of a highactivity promoter, a dicistronic vector or by gene amplification of thevector.

The nucleic acids in accordance with the invention can be expressed, oroverexpressed, in any in vitro or in vivo expression system. Any cellculture systems can be employed to express, or over-express, recombinantprotein, including bacterial, insect, yeast, fungal or mammaliancultures. Over-expression can be effected by appropriate choice ofpromoters, enhancers, vectors (such as use of replicon vectors,dicistronic vectors (see Gurtu (1996) Biochem. Biophys. Res. Commun229:295-8), media, culture systems and the like. In some embodiments,gene amplification using selection markers, such as glutamine synthetase(see Sanders (1987) Dev. Biol. Stand. 66:55-63), in cell systems areused to overexpress the polypeptides in accordance with the invention.

Additional details regarding this approach are in the public literatureand/or are known to the skilled artisan. In a particular non-limitingexemplification, such publicly available literature includes EP 0659215(WO 9403612 A1) (Nevalainen et al.); Lapidot, A., Mechaly, A., Shoham,Y., “Overexpression and single-step purification of a thermostablexylanase from Bacillus stearothermophilus T-6,” J. Biotechnol. November51:259-64 (1996); Lüthi, E., Jasmat, N. B., Bergquist, P. L., “Xylanasefrom the extremely thermophilic bacterium Caldocellum saccharolyticum:overexpression of the gene in Escherichia coli and characterization ofthe gene product,” Appl. Environ. Microbiol. September 56:2677-83(1990); and Sung, W. L., Luk, C. K., Zahab, D. M., Wakarchuk, W.,“Overexpression of the Bacillus subtilis and circulans xylanases inEscherichia coli,” Protein Expr. Purif. June 4:200-6 (1993), althoughthese references do not teach the inventive enzymes of the instantapplication.

The host cell may be any of the host cells familiar to those skilled inthe art, including prokaryotic cells, eukaryotic cells, mammalian cells,insect cells, or plant cells. As representative examples of appropriatehosts, there may be mentioned: bacterial cells, such as E. coli,Streptomyces, Bacillus subtilis, Bacillus cereus, Salmonella typhimuriumand various species within the genera Pseudomonas, Streptomyces andStaphylococcus, fungal cells, such as Aspergillus, yeast such as anyspecies of Pichia, Saccharomyces, Schizosaccharomyces, Schwanniomyces,including Pichia pastoris, Saccharomyces cerevisiae, orSchizosaccharomyces pombe, insect cells such as Drosophila S2 andSpodoptera Sf9, animal cells such as CHO, COS or Bowes melanoma andadenoviruses. The selection of an appropriate host is within theabilities of those skilled in the art.

The vector may be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes in accordancewith the invention. Following transformation of a suitable host strainand growth of the host strain to an appropriate cell density, theselected promoter may be induced by appropriate means (such astemperature shift or chemical induction) and the cells may be culturedfor an additional period to allow them to produce the desiredpolypeptide or fragment thereof.

Cells can be harvested by centrifugation, disrupted by physical orchemical means and the resulting crude extract is retained for furtherpurification. Microbial cells employed for expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents. Suchmethods are well known to those skilled in the art. The expressedpolypeptide or fragment thereof can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the polypeptide. If desired,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts (described by Gluzman,Cell, 23:175, 1981) and other cell lines capable of expressing proteinsfrom a compatible vector, such as the C127, 3T3, CHO, HeLa and BHK celllines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides in accordance withthe invention may or may not also include an initial methionine aminoacid residue.

Alternatively, the polypeptides in accordance with the invention, orfragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, or 150 or more consecutive amino acids thereof can be syntheticallyproduced by conventional peptide synthesizers, such as discussed below.In other embodiments, fragments or portions of the polypeptides may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides.

Cell-free translation systems can also be employed to produce one of thepolypeptides in accordance with the invention, or fragments comprisingat least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 or moreconsecutive amino acids thereof using mRNAs transcribed from a DNAconstruct comprising a promoter operably linked to a nucleic acidencoding the polypeptide or fragment thereof. In some embodiments, theDNA construct may be linearized prior to conducting an in vitrotranscription reaction. The transcribed mRNA is then incubated with anappropriate cell-free translation extract, such as a rabbit reticulocyteextract, to produce the desired polypeptide or fragment thereof.

Amplification of Nucleic Acids

In practicing the invention, nucleic acids in accordance with theinvention and nucleic acids encoding the aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzymes in accordance with theinvention, or modified nucleic acids in accordance with the invention,can be reproduced by amplification, such as PCR. Amplification can alsobe used to clone or modify the nucleic acids in accordance with theinvention. Thus, the invention provides amplification primer sequencepairs for amplifying nucleic acids in accordance with the invention. Oneof skill in the art can design amplification primer sequence pairs forany part of or the full length of these sequences.

In some embodiments, the invention provides nucleic acids amplified byamplification primer pairs in accordance with the invention, such asprimer pairs as set forth by about the first (the 5′) 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more residues of nucleicacids in accordance with the invention, and about the first (the 5′) 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more residues of thecomplementary strands. In some embodiments, the invention providesamplification primer sequence pairs for amplifying a nucleic acidencoding a polypeptide having an aldolase, such as pyruvate aldolase,such as HMG and/or KHG aldolase enzyme, activity, wherein the primerpair is capable of amplifying a nucleic acid comprising a sequence inaccordance with the invention, or fragments or subsequences thereof. Oneor each member of the amplification primer sequence pair can comprise anoligonucleotide comprising at least about 10 to 50 or more consecutivebases of the sequence, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25 or more consecutive bases of the sequence. In someembodiments, the invention provides amplification primer pairs, whereinthe primer pair comprises a first member having a sequence as set forthby about the first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 or more residues of a nucleic acid in accordance with theinvention, and a second member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,or 25 or more residues of the complementary strand of the first member.

The invention provides aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzymes generated by amplification, such aspolymerase chain reaction (PCR), using an amplification primer pair inaccordance with the invention. In some embodiments, the inventionprovides methods of making an aldolase, such as pyruvate aldolase, HMGand/or KHG aldolase enzyme by amplification, such as PCR, using anamplification primer pair in accordance with the invention. In someembodiments, the amplification primer pair amplifies a nucleic acid froma library, such as a gene library, such as an environmental library.

Amplification reactions can also be used to quantify the amount ofnucleic acid in a sample (such as the amount of message in a cellsample), label the nucleic acid (such as to apply it to an array or ablot), detect the nucleic acid, or quantify the amount of a specificnucleic acid in a sample. In some embodiments of the invention, messageisolated from a cell or a cDNA library are amplified.

The skilled artisan can select and design suitable oligonucleotideamplification primers. Amplification methods are also well known in theart, and include, such as polymerase chain reaction, PCR (see PCRPROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, AcademicPress, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press,Inc., N.Y., ligase chain reaction (LCR) (see Wu (1989) Genomics 4:560;Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);transcription amplification (see Kwoh (1989) Proc. Natl. Acad. Sci. USA86:1173); and, self-sustained sequence replication (see Guatelli (1990)Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicase amplification (seeSmith (1997) J. Clin. Microbiol. 35:1477-1491), automated Q-betareplicase amplification assay (see Burg (1996) Mol. Cell. Probes10:257-271) and other RNA polymerase mediated techniques (such as NASBA,Cangene, Mississauga, Ontario); see also Berger (1987) Methods Enzymol.152:307-316; Ausubel et al. Current Protocols in Molecular Biology, JohnWiley & Sons, Inc. 1997 and Sambrook et al., Molecular Cloning: ALaboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press (1989).U.S. Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology13:563-564.

Determining Sequence Identity in Nucleic Acids and Polypeptides

The invention provides nucleic acids comprising sequences having atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete(100%) sequence identity (homology) to a nucleic acid in accordance withthe invention (see Sequence Listing) over a region of at least about 50,75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1550 or more, residues. In some embodiments, theinvention provides polypeptides comprising sequences having at leastabout 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)sequence identity to a polypeptide in accordance with the invention (seeSequence Listing). The extent of sequence identity (homology) may bedetermined using any computer program and associated parameters,including those described herein, such as BLAST 2.2.2. or FASTA version3.0t78, with the default parameters.

Nucleic acid sequences in accordance with the invention can comprise atleast 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or500 or more consecutive nucleotides of a sequence in accordance with theinvention and sequences substantially identical thereto. Homologoussequences and fragments of nucleic acid sequences in accordance with theinvention can refer to a sequence having at least about 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity (homology) to thesesequences. Homology (sequence identity) may be determined using any ofthe computer programs and parameters described herein, including FASTAversion 3.0t78 with the default parameters. Homologous sequences alsoinclude RNA sequences in which uridines replace the thymines in thenucleic acid sequences in accordance with the invention. The homologoussequences may be obtained using any of the procedures described hereinor may result from the correction of a sequencing error. It will beappreciated that the nucleic acid sequences in accordance with theinvention can be represented in the traditional single character format(See the inside back cover of Stryer, Lubert. Biochemistry, 3rd Ed., W.H Freeman & Co., New York.) or in any other format which records theidentity of the nucleotides in a sequence.

In some embodiments, sequence comparison programs identified herein areused in this aspect in accordance with the invention, i.e., to determineif a nucleic acid or polypeptide sequence is within the scope inaccordance with the invention. However, protein and/or nucleic acidsequence identities (homologies) may be evaluated using any sequencecomparison algorithm or program known in the art. Such algorithms andprograms include, but are by no means limited to, TBLASTN, BLASTP,FASTA, TFASTA and CLUSTALW (see Pearson and Lipman, Proc. Natl. Acad.Sci. USA 85(8):2444-2448, 1988; Altschul et al., J. Mol. Biol.215(3):403-410, 1990; Thompson Nucleic Acids Res. 22(2):4673-4680, 1994;Higgins et al., Methods Enzymol. 266:383-402, 1996; Altschul et al., J.Mol. Biol. 215(3):403-410, 1990; Altschul et al., Nature Genetics3:266-272, 1993).

In some embodiments, homology or identity is measured using sequenceanalysis software (such as Sequence Analysis Software Package of theGenetics Computer Group, University of Wisconsin Biotechnology Center,1710 University Avenue, Madison, Wis. 53705). Such software matchessimilar sequences by assigning degrees of homology to various deletions,substitutions and other modifications. In some embodiments, the terms“homology” and “identity” in the context of two or more nucleic acids orpolypeptide sequences, refer to two or more sequences or subsequencesthat are the same or have a specified percentage of amino acid residuesor nucleotides that are the same when compared and aligned for maximumcorrespondence over a comparison window or designated region as measuredusing any number of sequence comparison algorithms or by manualalignment and visual inspection. In some embodiments, for sequencecomparison, one sequence acts as a reference sequence, to which testsequences are compared. When using a sequence comparison algorithm, testand reference sequences are entered into a computer, subsequencecoordinates are designated, if necessary and sequence algorithm programparameters are designated. Default program parameters can be used, oralternative parameters can be designated. The sequence comparisonalgorithm then calculates the percent sequence identities for the testsequences relative to the reference sequence, based on the programparameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencefor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, such as by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970,by the search for similarity method of person & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444, 1988, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection. Other algorithmsfor determining homology or identity include, for example, in additionto a BLAST program (Basic Local Alignment Search Tool at the NationalCenter for Biological Information), ALIGN, AMAS (Analysis of MultiplyAligned Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET(Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN(Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProvedSearcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W,CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, LasVegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign,Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence AnalysisPackage), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP(Local Content Program), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN,PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (SequenceAlignment by Genetic Algorithm) and WHAT-IF. Such alignment programs canalso be used to screen genome databases to identify polynucleotidesequences having substantially identical sequences. A number of genomedatabases are available, for example, a substantial portion of the humangenome is available as part of the Human Genome Sequencing Project(Gibbs, 1995). At least twenty-one other genomes have already beensequenced, including, for example, M. genitalium (Fraser et al., Science270:397-403 (1995)), M. jannaschii (Bult et al., Science 23:1058-73(1996)), H. influenzae (Fleischmann et al., Science 269:496-512 (1995)),E. coli (Blattner et al., Science 277:1453-74 (1997)) and yeast (S.cerevisiae) (Mewes et al., Nature 387:7-65 (1997)) and D. melanogaster(Adams et al., Science 287:2185-95 (2000)). Significant progress hasalso been made in sequencing the genomes of model organism, such asmouse, C. elegans and Arabadopsis sp. Several databases containinggenomic information annotated with some functional information aremaintained by different organizations and may be accessible via theinternet.

In some embodiments, BLAST and BLAST 2.0 algorithms are used, which aredescribed in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977 andAltschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T and X determinethe sensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3 and expectations (E) of 10 and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see Karlin & Altschul, Proc. Natl.Acad. Sci. USA 90:5873, 1993). One measure of similarity provided byBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a nucleicacid is considered similar to a references sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.2, n some embodiments less than about0.01 and in other embodiments less than about 0.001.

In some embodiments, protein and nucleic acid sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”) Inparticular, five specific BLAST programs are used to perform thefollowing task:

(1) BLASTP and BLAST3 compare an amino acid query sequence against aprotein sequence database;

(2) BLASTN compares a nucleotide query sequence against a nucleotidesequence database;

(3) BLASTX compares the six-frame conceptual translation products of aquery nucleotide sequence (both strands) against a protein sequencedatabase;

(4) TBLASTN compares a query protein sequence against a nucleotidesequence database translated in all six reading frames (both strands);and

(5) TBLASTX compares the six-frame translations of a nucleotide querysequence against the six-frame translations of a nucleotide sequencedatabase.

The BLAST programs identify homologous sequences by identifying similarsegments, which are referred to herein as “high-scoring segment pairs,”between a query amino or nucleic acid sequence and a test sequence whichis, in some embodiments, obtained from a protein or nucleic acidsequence database. High-scoring segment pairs are, in some embodiments,identified (i.e., aligned) by means of a scoring matrix, many of whichare known in the art. In some embodiments, the scoring matrix used isthe BLOSUM62 matrix (Gonnet (1992) Science 256:1443-1445; Henikoff andHenikoff (1993) Proteins 17:49-61). Less In some embodiments, the PAM orPAM250 matrices may also be used (see Schwartz and Dayhoff, eds., 1978,Matrices for Detecting Distance Relationships: Atlas of Protein Sequenceand Structure, Washington: National Biomedical Research Foundation).BLAST programs are accessible through the U.S. National Library ofMedicine.

The parameters used with the above algorithms may be adapted dependingon the sequence length and degree of homology studied. In someembodiments, the parameters may be the default parameters used by thealgorithms in the absence of instructions from the user.

Computer Systems and Computer Program Products

The invention provides computers, computer systems, computer readablemediums, computer programs products and the like recorded or storedthereon the nucleic acid and polypeptide sequences in accordance withthe invention. Additionally, in practicing the methods in accordancewith the invention, such as to determine and identify sequenceidentities (to determine whether a nucleic acid is within the scope inaccordance with the invention), structural homologies, motifs and thelike in silico, a nucleic acid or polypeptide sequence in accordancewith the invention can be stored, recorded, and manipulated on anymedium which can be read and accessed by a computer.

As used herein, the words “recorded” and “stored” refer to a process forstoring information on a computer medium. A skilled artisan can readilyadopt any known methods for recording information on a computer readablemedium to generate manufactures comprising one or more of the nucleicacid and/or polypeptide sequences in accordance with the invention. Asused herein, the terms “computer,” “computer program” and “processor”are used in their broadest general contexts and incorporate all suchdevices, as described in detail, below. A “coding sequence of” or a“sequence encodes” a particular polypeptide or protein, is a nucleicacid sequence which is transcribed and translated into a polypeptide orprotein when placed under the control of appropriate regulatorysequences.

The polypeptides in accordance with the invention include sequences inaccordance with the invention and sequences substantially identicalthereto, and subsequences and enzymatically active fragments of any ofthe preceding sequences. In some embodiments, substantially identical,or homologous, polypeptide sequences refer to a polypeptide sequencehaving at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, orcomplete (100%) sequence identity (homology) to sequence in accordancewith the invention.

Homology (sequence identity) may be determined using any of the computerprograms and parameters described herein. A nucleic acid or polypeptidesequence in accordance with the invention can be stored, recorded andmanipulated on any medium which can be read and accessed by a computer.As used herein, the words “recorded” and “stored” refer to a process forstoring information on a computer medium. A skilled artisan can readilyadopt any of the presently known methods for recording information on acomputer readable medium to generate manufactures comprising one or moreof the nucleic acid sequences in accordance with the invention, one ormore of the polypeptide sequences in accordance with the invention.Another embodiment of the invention is a computer readable medium havingrecorded thereon at least 2, 5, 10, 15, or 20 or more nucleic acid orpolypeptide sequences in accordance with the invention.

Another embodiment of the invention is a computer readable medium havingrecorded thereon one or more of the nucleic acid sequences in accordancewith the invention. Another embodiment of the invention is a computerreadable medium having recorded thereon one or more of the polypeptidesequences in accordance with the invention. Another embodiment of theinvention is a computer readable medium having recorded thereon at least2, 5, 10, 15, or 20 or more of the nucleic acid or polypeptide sequencesas set forth above.

Computer readable media include magnetically readable media, opticallyreadable media, electronically readable media and magnetic/opticalmedia. For example, the computer readable media may be a hard disk, afloppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD),Random Access Memory (RAM), or Read Only Memory (ROM) as well as othertypes of other media known to those skilled in the art.

Some embodiments of the invention include systems (such as internetbased systems), such as computer systems which store and manipulate thesequence information described herein. One example of a computer system100 is illustrated in block diagram form in FIG. 9. As used herein, “acomputer system” refers to the hardware components, software componentsand data storage components used to analyze a nucleotide sequence of anucleic acid sequence in accordance with the invention, or a polypeptidesequence in accordance with the invention. In some embodiments, thecomputer system 100 includes a processor for processing, accessing andmanipulating the sequence data. The processor 105 can be any well-knowntype of central processing unit, such as, for example, the Pentium IIIfrom Intel Corporation, or similar processor from Sun, Motorola, Compaq,AMD or International Business Machines.

In some embodiments, the computer system 100 is a general purpose systemthat comprises the processor 105 and one or more internal data storagecomponents 110 for storing data and one or more data retrieving devicesfor retrieving the data stored on the data storage components. A skilledartisan can readily appreciate that any one of the currently availablecomputer systems are suitable.

In one embodiment, the computer system 100 includes a processor 105connected to a bus which is connected to a main memory 115 (in oneembodiment implemented as RAM) and one or more internal data storagedevices 110, such as a hard drive and/or other computer readable mediahaving data recorded thereon. In some embodiments, the computer system100 further includes one or more data retrieving device 118 for readingthe data stored on the internal data storage devices 110.

The data retrieving device 118 may represent, for example, a floppy diskdrive, a compact disk drive, a magnetic tape drive, or a modem capableof connection to a remote data storage system (such as via the internet)etc. In some embodiments, the internal data storage device 110 is aremovable computer readable medium such as a floppy disk, a compactdisk, a magnetic tape, etc. containing control logic and/or datarecorded thereon. The computer system 100 may advantageously include orbe programmed by appropriate software for reading the control logicand/or the data from the data storage component once inserted in thedata retrieving device.

The computer system 100 includes a display 120 which is used to displayoutput to a computer user. It should also be noted that the computersystem 100 can be linked to other computer systems 125 a-c in a networkor wide area network to provide centralized access to the computersystem 100.

Software for accessing and processing the nucleotide sequences of anucleic acid sequence in accordance with the invention and sequencessubstantially identical thereto, or a polypeptide sequence in accordancewith the invention and sequences substantially identical thereto, (suchas search tools, compare tools and modeling tools etc.) may reside inmain memory 115 during execution.

In some embodiments, the computer system 100 may further comprise asequence comparison algorithm for comparing a nucleic acid sequence inaccordance with the invention and sequences substantially identicalthereto, or a polypeptide sequence in accordance with the invention andsequences substantially identical thereto, stored on a computer readablemedium to a reference nucleotide or polypeptide sequence(s) stored on acomputer readable medium. A “sequence comparison algorithm” refers toone or more programs which are implemented (locally or remotely) on thecomputer system 100 to compare a nucleotide sequence with othernucleotide sequences and/or compounds stored within a data storagemeans. For example, the sequence comparison algorithm may compare thenucleotide sequences of a nucleic acid sequence in accordance with theinvention and sequences substantially identical thereto, or apolypeptide sequence in accordance with the invention and sequencessubstantially identical thereto, stored on a computer readable medium toreference sequences stored on a computer readable medium to identifyhomologies or structural motifs.

FIG. 10 is a flow diagram illustrating one embodiment of a process 200for comparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database. The database of sequencescan be a private database stored within the computer system 100, or apublic database such as GENBANK that is available through the Internet.

The process 200 begins at a start state 201 and then moves to a state202 wherein the new sequence to be compared is stored to a memory in acomputer system 100. As discussed above, the memory could be any type ofmemory, including RAM or an internal storage device.

The process 200 then moves to a state 204 wherein a database ofsequences is opened for analysis and comparison. The process 200 thenmoves to a state 206 wherein the first sequence stored in the databaseis read into a memory on the computer. A comparison is then performed ata state 210 to determine if the first sequence is the same as the secondsequence. It is important to note that this step is not limited toperforming an exact comparison between the new sequence and the firstsequence in the database. Well-known methods are known to those of skillin the art for comparing two nucleotide or protein sequences, even ifthey are not identical. For example, gaps can be introduced into onesequence in order to raise the homology level between the two testedsequences. The parameters that control whether gaps or other featuresare introduced into a sequence during comparison are normally entered bythe user of the computer system.

Once a comparison of the two sequences has been performed at the state210, a determination is made at a decision state 210 whether the twosequences are the same. Of course, the term “same” is not limited tosequences that are absolutely identical. Sequences that are within thehomology parameters entered by the user will be marked as “same” in theprocess 200.

If a determination is made that the two sequences are the same, theprocess 200 moves to a state 214 wherein the name of the sequence fromthe database is displayed to the user. This state notifies the user thatthe sequence with the displayed name fulfills the homology constraintsthat were entered. Once the name of the stored sequence is displayed tothe user, the process 200 moves to a decision state 218 wherein adetermination is made whether more sequences exist in the database. Ifno more sequences exist in the database, then the process 200 terminatesat an end state 220. However, if more sequences do exist in thedatabase, then the process 200 moves to a state 224 wherein a pointer ismoved to the next sequence in the database so that it can be compared tothe new sequence. In this manner, the new sequence is aligned andcompared with every sequence in the database.

It should be noted that if a determination had been made at the decisionstate 212 that the sequences were not homologous, then the process 200would move immediately to the decision state 218 in order to determineif any other sequences were available in the database for comparison.

Accordingly, one embodiment of the invention is a computer systemcomprising a processor, a data storage device having stored thereon anucleic acid sequence in accordance with the invention and sequencessubstantially identical thereto, or a polypeptide sequence in accordancewith the invention and sequences substantially identical thereto, a datastorage device having retrievably stored thereon reference nucleotidesequences or polypeptide sequences to be compared to a nucleic acidsequence in accordance with the invention and sequences substantiallyidentical thereto, or a polypeptide sequence in accordance with theinvention and sequences substantially identical thereto and a sequencecomparer for conducting the comparison. The sequence comparer mayindicate a homology level between the sequences compared or identifystructural motifs in the above described nucleic acid code a nucleicacid sequence in accordance with the invention and sequencessubstantially identical thereto, or a polypeptide sequence in accordancewith the invention and sequences substantially identical thereto, or itmay identify structural motifs in sequences which are compared to thesenucleic acid codes and polypeptide codes. In some embodiments, the datastorage device may have stored thereon the sequences of at least 2, 5,10, 15, 20, 25, 30 or 40 or more of the nucleic acid sequences inaccordance with the invention and sequences substantially identicalthereto, or the polypeptide sequences in accordance with the inventionand sequences substantially identical thereto.

Another embodiment of the invention is a method for determining thelevel of homology between a nucleic acid sequence in accordance with theinvention and sequences substantially identical thereto, or apolypeptide sequence in accordance with the invention and sequencessubstantially identical thereto and a reference nucleotide sequence. Themethod including reading the nucleic acid code or the polypeptide codeand the reference nucleotide or polypeptide sequence through the use ofa computer program which determines homology levels and determininghomology between the nucleic acid code or polypeptide code and thereference nucleotide or polypeptide sequence with the computer program.The computer program may be any of a number of computer programs fordetermining homology levels, including those specifically enumeratedherein, (such as BLAST2N with the default parameters or with anymodified parameters). The method may be implemented using the computersystems described above. The method may also be performed by reading atleast 2, 5, 10, 15, 20, 25, 30 or 40 or more of the above describednucleic acid sequences in accordance with the invention and sequencessubstantially identical thereto, or the polypeptide sequences inaccordance with the invention and sequences substantially identicalthereto through use of the computer program and determining homologybetween the nucleic acid codes or polypeptide codes and referencenucleotide sequences or polypeptide sequences.

FIG. 11 is a flow diagram illustrating one embodiment of a process 250in a computer for determining whether two sequences are homologous. Theprocess 250 begins at a start state 252 and then moves to a state 254wherein a first sequence to be compared is stored to a memory. Thesecond sequence to be compared is then stored to a memory at a state256. The process 250 then moves to a state 260 wherein the firstcharacter in the first sequence is read and then to a state 262 whereinthe first character of the second sequence is read. It should beunderstood that if the sequence is a nucleotide sequence, then thecharacter would normally be either A, T, C, G or U. If the sequence is aprotein sequence, then it is, in some embodiments, in the single letteramino acid code so that the first and sequence sequences can be easilycompared.

A determination is then made at a decision state 264 whether the twocharacters are the same. If they are the same, then the process 250moves to a state 268 wherein the next characters in the first and secondsequences are read. A determination is then made whether the nextcharacters are the same. If they are, then the process 250 continuesthis loop until two characters are not the same. If a determination ismade that the next two characters are not the same, the process 250moves to a decision state 274 to determine whether there are any morecharacters either sequence to read.

If there are not any more characters to read, then the process 250 movesto a state 276 wherein the level of homology between the first andsecond sequences is displayed to the user. The level of homology isdetermined by calculating the proportion of characters between thesequences that were the same out of the total number of sequences in thefirst sequence. Thus, if every character in a first 100 nucleotidesequence aligned with a every character in a second sequence, thehomology level would be 100%.

Alternatively, the computer program may be a computer program whichcompares the nucleotide sequences of a nucleic acid sequence as setforth in the invention, to one or more reference nucleotide sequences inorder to determine whether the nucleic acid code in accordance with theinvention and sequences substantially identical thereto, differs from areference nucleic acid sequence at one or more positions. Optionallysuch a program records the length and identity of inserted, deleted orsubstituted nucleotides with respect to the sequence of either thereference polynucleotide or a nucleic acid sequence in accordance withthe invention and sequences substantially identical thereto. In someembodiments, the computer program may be a program which determineswhether a nucleic acid sequence in accordance with the invention andsequences substantially identical thereto, contains a single nucleotidepolymorphism (SNP) with respect to a reference nucleotide sequence.

Accordingly, another embodiment of the invention is a method fordetermining whether a nucleic acid sequence in accordance with theinvention and sequences substantially identical thereto, differs at oneor more nucleotides from a reference nucleotide sequence comprising thesteps of reading the nucleic acid code and the reference nucleotidesequence through use of a computer program which identifies differencesbetween nucleic acid sequences and identifying differences between thenucleic acid code and the reference nucleotide sequence with thecomputer program. In some embodiments, the computer program is a programwhich identifies single nucleotide polymorphisms. The method may beimplemented by the computer systems described above and the methodillustrated in FIG. 11. The method may also be performed by reading atleast 2, 5, 10, 15, 20, 25, 30, or 40 or more of the nucleic acidsequences in accordance with the invention and sequences substantiallyidentical thereto and the reference nucleotide sequences through the useof the computer program and identifying differences between the nucleicacid codes and the reference nucleotide sequences with the computerprogram.

In other embodiments, the computer based system may further comprise anidentifier for identifying features within a nucleic acid sequence inaccordance with the invention or a polypeptide sequence in accordancewith the invention and sequences substantially identical thereto. An“identifier” refers to one or more programs which identifies certainfeatures within a nucleic acid sequence in accordance with theinvention, or a polypeptide sequence in accordance with the invention.In some embodiments, the identifier may comprise a program whichidentifies an open reading frame in a nucleic acid sequence inaccordance with the invention and sequences substantially identicalthereto.

FIG. 12 is a flow diagram illustrating one embodiment of an identifierprocess 300 for detecting the presence of a feature in a sequence. Theprocess 300 begins at a start state 302 and then moves to a state 304wherein a first sequence that is to be checked for features is stored toa memory 115 in the computer system 100. The process 300 then moves to astate 306 wherein a database of sequence features is opened. Such adatabase would include a list of each feature's attributes along withthe name of the feature. For example, a feature name could be“Initiation Codon” and the attribute would be “ATG”. Another examplewould be the feature name “TAATAA Box” and the feature attribute wouldbe “TAATAA”. An example of such a database is produced by the Universityof Wisconsin Genetics Computer Group. Alternatively, the features may bestructural polypeptide motifs such as alpha helices, beta sheets, orfunctional polypeptide motifs such as enzymatic active sites,helix-turn-helix motifs or other motifs known to those skilled in theart.

Once the database of features is opened at the state 306, the process300 moves to a state 308 wherein the first feature is read from thedatabase. A comparison of the attribute of the first feature with thefirst sequence is then made at a state 310. A determination is then madeat a decision state 316 whether the attribute of the feature was foundin the first sequence. If the attribute was found, then the process 300moves to a state 318 wherein the name of the found feature is displayedto the user.

The process 300 then moves to a decision state 320 wherein adetermination is made whether move features exist in the database. If nomore features do exist, then the process 300 terminates at an end state324. However, if more features do exist in the database, then theprocess 300 reads the next sequence feature at a state 326 and loopsback to the state 310 wherein the attribute of the next feature iscompared against the first sequence. It should be noted, that if thefeature attribute is not found in the first sequence at the decisionstate 316, the process 300 moves directly to the decision state 320 inorder to determine if any more features exist in the database.

Accordingly, another embodiment of the invention is a method ofidentifying a feature within a nucleic acid sequence in accordance withthe invention and sequences substantially identical thereto, or apolypeptide sequence in accordance with the invention and sequencessubstantially identical thereto, comprising reading the nucleic acidcode(s) or polypeptide code(s) through the use of a computer programwhich identifies features therein and identifying features within thenucleic acid code(s) with the computer program. In some embodiments, thecomputer program comprises a computer program which identifies openreading frames. The method may be performed by reading a single sequenceor at least 2, 5, 10, 15, 20, 25, 30, or 40 or more of the nucleic acidsequences in accordance with the invention and sequences substantiallyidentical thereto, or the polypeptide sequences in accordance with theinvention and sequences substantially identical thereto, through the useof the computer program and identifying features within the nucleic acidcodes or polypeptide codes with the computer program.

A nucleic acid sequence in accordance with the invention and sequencessubstantially identical thereto, or a polypeptide sequence in accordancewith the invention and sequences substantially identical thereto, may bestored and manipulated in a variety of data processor programs in avariety of formats. For example, a nucleic acid sequence in accordancewith the invention and sequences substantially identical thereto, or apolypeptide sequence in accordance with the invention and sequencessubstantially identical thereto, may be stored as text in a wordprocessing file, such as Microsoft WORD™ or WORDPERFECT™ or as an ASCIIfile in a variety of database programs familiar to those of skill in theart, such as DB2™, SYBASE™, or ORACLE™. In addition, many computerprograms and databases may be used as sequence comparison algorithms,identifiers, or sources of reference nucleotide sequences or polypeptidesequences to be compared to a nucleic acid sequence in accordance withthe invention and sequences substantially identical thereto, or apolypeptide sequence in accordance with the invention and sequencessubstantially identical thereto. The following list is intended not tolimit the invention but to provide guidance to programs and databaseswhich are useful with the nucleic acid sequences in accordance with theinvention and sequences substantially identical thereto, or thepolypeptide sequences in accordance with the invention and sequencessubstantially identical thereto.

The programs and databases which may be used include, but are notlimited to: MACPATTERN™ (EMBL), DISCOVERYBASE™ (Molecular applicationsGroup), GENEMINE™ (Molecular applications Group), LOOK™ (Molecularapplications Group), MACLOOK™ (Molecular applications Group), BLAST andBLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol. Biol. 215:403, 1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444, 1988), FASTDB (Brutlag et al. Comp. App. Biosci. 6:237-245, 1990),CATALYST™ (Molecular Simulations Inc.), Catalyst/SHAPE™ (MolecularSimulations Inc.), Cerius².DBAccess™ (Molecular Simulations Inc.),HYPOGEN™ (Molecular Simulations Inc.), INSIGHT II™, (MolecularSimulations Inc.), DISCOVER™ (Molecular Simulations Inc.), CHARMm™(Molecular Simulations Inc.), FELIX™ (Molecular Simulations Inc.),DELPHI™ (Molecular Simulations Inc.), QuanteMM™, (Molecular SimulationsInc.), Homology (Molecular Simulations Inc.), MODELER™ (MolecularSimulations Inc.), ISIS™ (Molecular Simulations Inc.), Quanta/ProteinDesign (Molecular Simulations Inc.), WebLab (Molecular SimulationsInc.), WebLab Diversity Explorer (Molecular Simulations Inc.), GeneExplorer (Molecular Simulations Inc.), SeqFold (Molecular SimulationsInc.), the MDL Available Chemicals Directory database, the MDL Drug DataReport data base, the Comprehensive Medicinal Chemistry database,Derwents's World Drug Index database, the BioByteMasterFile database,the Genbank database and the Genseqn database. Many other programs anddata bases would be apparent to one of skill in the art given thepresent disclosure.

Motifs which may be detected using the above programs include sequencesencoding leucine zippers, helix-turn-helix motifs, glycosylation sites,ubiquitination sites, alpha helices and beta sheets, signal sequencesencoding signal peptides which direct the secretion of the encodedproteins, sequences implicated in transcription regulation such ashomeoboxes, acidic stretches, enzymatic active sites, substrate bindingsites and enzymatic cleavage sites.

Hybridization of Nucleic Acids

The invention provides isolated, synthetic or recombinant nucleic acidsthat hybridize under stringent conditions to a sequence in accordancewith the invention (such as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ IDNO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ IDNO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ IDNO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ IDNO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125,SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ IDNO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153,SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ IDNO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181,SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ IDNO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209,SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ IDNO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237,SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ IDNO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265,SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ ID NO:273, SEQ IDNO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQID NO:285, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293,SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ IDNO:303, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321,SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ IDNO:331, SEQ ID NO:333, SEQ ID NO:335, SEQ ID NO:336, SEQ ID NO:337, orSEQ ID NO:338. The stringent conditions can be highly stringentconditions, medium stringent conditions and/or low stringent conditions,including the high and reduced stringency conditions described herein.In some embodiments, it is the stringency of the wash conditions thatset forth the conditions which determine whether a nucleic acid iswithin the scope in accordance with the invention, as discussed below.

“Hybridization” refers to the process by which a nucleic acid strandjoins with a complementary strand through base pairing. Hybridizationreactions can be sensitive and selective so that a particular sequenceof interest can be identified even in samples in which it is present atlow concentrations. Suitably stringent conditions can be defined by, forexample, the concentrations of salt or formamide in the prehybridizationand hybridization solutions, or by the hybridization temperature and arewell known in the art. In other embodiments, stringency can be increasedby reducing the concentration of salt, increasing the concentration offormamide, or raising the hybridization temperature. In otherembodiments, nucleic acids in accordance with the invention are definedby their ability to hybridize under various stringency conditions (suchas high, medium, and low), as set forth herein.

In some embodiments, hybridization under high stringency conditionscomprise about 50% formamide at about 37° C. to 42° C. In someembodiments, hybridization conditions comprise reduced stringencyconditions in about 35% to 25% formamide at about 30° C. to 35° C. Insome embodiments, hybridization conditions comprise high stringencyconditions, such as at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS and 200μg/ml sheared and denatured salmon sperm DNA. In some embodiments,hybridization conditions comprise these reduced stringency conditions,but in 35% formamide at a reduced temperature of 35° C. The temperaturerange corresponding to a particular level of stringency can be furthernarrowed by calculating the purine to pyrimidine ratio of the nucleicacid of interest and adjusting the temperature accordingly. Variationson the above ranges and conditions are well known in the art.

In other embodiments, nucleic acids in accordance with the invention asdefined by their ability to hybridize under stringent conditions can bebetween about five residues and the full length of nucleic acid inaccordance with the invention; such as they can be at least 5, 10, 15,20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, or more, residues in length. Nucleic acids shorter than fulllength are also included. These nucleic acids can be useful as, such ashybridization probes, labeling probes, PCR oligonucleotide probes, siRNAor miRNA (single or double stranded), antisense or sequences encodingantibody binding peptides (epitopes), motifs, active sites and the like.

In some embodiments, nucleic acids in accordance with the invention aredefined by their ability to hybridize under high stringency comprisesconditions of about 50% formamide at about 37° C. to 42° C. In someembodiments, nucleic acids in accordance with the invention are definedby their ability to hybridize under reduced stringency comprisingconditions in about 35% to 25% formamide at about 30° C. to 35° C.

Alternatively, nucleic acids in accordance with the invention aredefined by their ability to hybridize under high stringency comprisingconditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS, and arepetitive sequence blocking nucleic acid, such as cot-1 or salmon spermDNA (such as 200 μg/ml sheared and denatured salmon sperm DNA). In someembodiments, nucleic acids in accordance with the invention are definedby their ability to hybridize under reduced stringency conditionscomprising 35% or 40% formamide at a reduced temperature of 35° C. or42° C.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (such as GC v. ATcontent) and nucleic acid type (such as RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

Hybridization may be carried out under conditions of low stringency,moderate stringency or high stringency. As an example of nucleic acidhybridization, a polymer membrane containing immobilized denaturednucleic acids is first prehybridized for 30 minutes at 45° C. in asolution consisting of 0.9 M NaCl, 50 mM NaH₂PO₄, pH 7.0, 5.0 mMNa₂EDTA, 0.5% SDS, 10×Denhardt's and 0.5 mg/ml polyriboadenylic acid.Approximately 2×10⁷ cpm (specific activity 4−9×10⁸ cpm/μg) of ³²Pend-labeled oligonucleotide probe are then added to the solution. After12-16 hours of incubation, the membrane is washed for 30 minutes at roomtemperature in 1×SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1mM Na₂EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh1×SET at T_(m)-10° C. for the oligonucleotide probe. The membrane isthen exposed to auto-radiographic film for detection of hybridizationsignals. All of the foregoing hybridizations would be considered to beunder conditions of high stringency.

Following hybridization, a filter can be washed to remove anynon-specifically bound detectable probe. The stringency used to wash thefilters can also be varied depending on the nature of the nucleic acidsbeing hybridized, the length of the nucleic acids being hybridized, thedegree of complementarity, the nucleotide sequence composition (such asGC v. AT content) and the nucleic acid type (such as RNA v. DNA).Examples of progressively higher stringency condition washes are asfollows: 2×SSC, 0.1% SDS at room temperature for 15 minutes (lowstringency); 0.1×SSC, 0.5% SDS at room temperature for 30 minutes to 1hour (moderate stringency); 0.1×SSC, 0.5% SDS for 15 to 30 minutes atbetween the hybridization temperature and 68° C. (high stringency); and0.15M NaCl for 15 minutes at 72° C. (very high stringency). A final lowstringency wash can be conducted in 0.1×SSC at room temperature. Theexamples above are merely illustrative of one set of conditions that canbe used to wash filters. One of skill in the art would know that thereare numerous recipes for different stringency washes. Some otherexamples are given below.

In some embodiments, hybridization conditions comprise a wash stepcomprising a wash for 30 minutes at room temperature in a solutioncomprising 1×150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mMNa₂EDTA, 0.5% SDS, followed by a 30 minute wash in fresh solution.

Nucleic acids which have hybridized to the probe are identified byautoradiography or other conventional techniques.

The above procedures may be modified to identify nucleic acids havingdecreasing levels of sequence identity (homology) to the probe sequence.For example, to obtain nucleic acids of decreasing sequence identity(homology) to the detectable probe, less stringent conditions may beused. For example, the hybridization temperature may be decreased inincrements of 5° C. from 68° C. to 42° C. in a hybridization bufferhaving a Na⁺ concentration of approximately 1M. Following hybridization,the filter may be washed with 2×SSC, 0.5% SDS at the temperature ofhybridization. These conditions are considered to be “moderate”conditions above 50° C. and “low” conditions below 50° C. A specificexample of “moderate” hybridization conditions is when the abovehybridization is conducted at 55° C. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 45° C.

Alternatively, the hybridization may be carried out in buffers, such as6×SSC, containing formamide at a temperature of 42° C. In this case, theconcentration of formamide in the hybridization buffer may be reduced in5% increments from 50% to 0% to identify clones having decreasing levelsof homology to the probe. Following hybridization, the filter may bewashed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered tobe “moderate” conditions above 25% formamide and “low” conditions below25% formamide. A specific example of “moderate” hybridization conditionsis when the above hybridization is conducted at 30% formamide. Aspecific example of “low stringency” hybridization conditions is whenthe above hybridization is conducted at 10% formamide.

However, the selection of a hybridization format may not be critical—itis the stringency of the wash conditions that set forth the conditionswhich determine whether a nucleic acid is within the scope in accordancewith the invention. Wash conditions used to identify nucleic acidswithin the scope in accordance with the invention include, such as: asalt concentration of about 0.02 molar at pH 7 and a temperature of atleast about 50° C. or about 55° C. to about 60° C.; or, a saltconcentration of about 0.15 M NaCl at 72° C. for about 15 minutes; or, asalt concentration of about 0.2×SSC at a temperature of at least about50° C. or about 55° C. to about 60° C. for about 15 to about 20 minutes;or, the hybridization complex is washed twice with a solution with asalt concentration of about 2×SSC containing 0.1% SDS at roomtemperature for 15 minutes and then washed twice by 0.1×SSC containing0.1% SDS at 68° C. for 15 minutes; or, equivalent conditions. SeeSambrook ed., MOLECULAR CLONING: A LABORATORY MANUAL (2nd ed.), vols.1-3, Cold Spring Harbor Laboratory (1989), LABORATORY TECHNIQUES INBIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACIDPROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed.Elsevier, N.Y. (1993) and Ausubel, ed. John Wiley & Sons, Inc., New York(1997) for a description of SSC buffer and equivalent conditions.

These methods may be used to isolate or identify nucleic acids inaccordance with the invention and sequences substantially identicalthereto. For example, the preceding methods may be used to isolate oridentify nucleic acids having a sequence with at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity (homology) to anucleic acid sequence selected from the group consisting of one of thesequences in accordance with the invention and sequences substantiallyidentical thereto, or fragments comprising at least about 10, 15, 20,25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutivebases thereof and the sequences complementary thereto. Sequence identity(homology) may be measured using the alignment algorithm. For example,the homologous polynucleotides may have a coding sequence which is anaturally occurring allelic variant of one of the coding sequencesdescribed herein. Such allelic variants may have a substitution,deletion or addition of one or more nucleotides when compared to thenucleic acids in accordance with the invention. Additionally, the aboveprocedures may be used to isolate nucleic acids which encodepolypeptides having at least about 99%, 95%, at least 90%, at least 85%,at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, atleast 55%, or at least 50% sequence identity (homology) to a polypeptidein accordance with the invention, or fragments comprising at least 5,10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof as determined using a sequence alignment algorithm (such as theFASTA version 3.0t78 algorithm with the default parameters).

Oligonucleotides Probes and Methods for Using them

The invention also provides nucleic acid probes that can be used, suchas for identifying, amplifying, or isolating nucleic acids encoding apolypeptide having an aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzyme activity or fragments thereof or foridentifying aldolase, such as pyruvate aldolase, such as HMG and/or KHGaldolase enzyme, genes. In some embodiments, the probe comprises atleast about 10 consecutive bases of a nucleic acid in accordance withthe invention. Alternatively, a probe in accordance with the inventioncan be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,110, 120, 130, 150 or about 10 to 50, about 20 to 60 about 30 to 70,consecutive bases of a sequence as set forth in a nucleic acid inaccordance with the invention. The probes identify a nucleic acid bybinding and/or hybridization. The probes can be used in arrays inaccordance with the invention, see discussion below, including, such ascapillary arrays. The probes in accordance with the invention can alsobe used to isolate other nucleic acids or polypeptides.

The isolated, synthetic or recombinant nucleic acids in accordance withthe invention, the sequences complementary thereto, or a fragmentcomprising at least about 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150,200, 300, 400, or 500 consecutive bases of one of the sequences inaccordance with the invention, or the sequences complementary theretomay also be used as probes to determine whether a biological sample,such as a soil sample, contains an organism having a nucleic acidsequence in accordance with the invention or an organism from which thenucleic acid was obtained. In such procedures, a biological samplepotentially harboring the organism from which the nucleic acid wasisolated is obtained and nucleic acids are obtained from the sample. Thenucleic acids are contacted with the probe under conditions which permitthe probe to specifically hybridize to any complementary sequences fromwhich are present therein.

Where necessary, conditions which permit the probe to specificallyhybridize to complementary sequences may be determined by placing theprobe in contact with complementary sequences from samples known tocontain the complementary sequence as well as control sequences which donot contain the complementary sequence. Hybridization conditions, suchas the salt concentration of the hybridization buffer, the formamideconcentration of the hybridization buffer, or the hybridizationtemperature, may be varied to identify conditions which allow the probeto hybridize specifically to complementary nucleic acids.

If the sample contains the organism from which the nucleic acid wasisolated, specific hybridization of the probe is then detected.Hybridization may be detected by labeling the probe with a detectableagent such as a radioactive isotope, a fluorescent dye or an enzymecapable of catalyzing the formation of a detectable product.

Many methods for using the labeled probes to detect the presence ofcomplementary nucleic acids in a sample are familiar to those skilled inthe art. These include Southern Blots, Northern Blots, colonyhybridization procedures and dot blots. Protocols for each of theseprocedures are provided in Ausubel et al. Current Protocols in MolecularBiology, John Wiley & Sons, Inc. (1997) and Sambrook et al., MolecularCloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor LaboratoryPress (1989).

Alternatively, more than one probe (at least one of which is capable ofspecifically hybridizing to any complementary sequences which arepresent in the nucleic acid sample), may be used in an amplificationreaction to determine whether the sample contains an organism containinga nucleic acid sequence in accordance with the invention (such as anorganism from which the nucleic acid was isolated). In some embodiments,the probes comprise oligonucleotides. In some embodiments, theamplification reaction may comprise a PCR reaction. PCR protocols aredescribed in Ausubel and Sambrook, supra. Alternatively, theamplification may comprise a ligase chain reaction, 3SR, or stranddisplacement reaction. (See Barany, F., “The Ligase Chain Reaction in aPCR World”, PCR Methods and applications 1:5-16, 1991; E. Fahy et al.,“Self-sustained Sequence Replication (3SR): An IsothermalTranscription-based Amplification System Alternative to PCR”, PCRMethods and applications 1:25-33, 1991; and Walker G. T. et al., “StrandDisplacement Amplification—an Isothermal in vitro DNA AmplificationTechnique”, Nucleic Acid Research 20:1691-1696, 1992). In suchprocedures, the nucleic acids in the sample are contacted with theprobes, the amplification reaction is performed and any resultingamplification product is detected. The amplification product may bedetected by performing gel electrophoresis on the reaction products andstaining the gel with an intercalator such as ethidium bromide.Alternatively, one or more of the probes may be labeled with aradioactive isotope and the presence of a radioactive amplificationproduct may be detected by autoradiography after gel electrophoresis.

Probes derived from sequences near the ends of the sequences inaccordance with the invention, may also be used in chromosome walkingprocedures to identify clones containing genomic sequences locatedadjacent to the sequences in accordance with the invention. Such methodsallow the isolation of genes which encode additional proteins from thehost organism.

In some embodiments, the isolated, synthetic or recombinant nucleicacids in accordance with the invention, the sequences complementarythereto, or a fragment comprising at least 10, 15, 20, 25, 30, 35, 40,50, 75, 100, 150, 200, 300, 400, or 500 or more consecutive bases of oneof the sequences in accordance with the invention, or the sequencescomplementary thereto are used as probes to identify and isolate relatednucleic acids. In some embodiments, the related nucleic acids may becDNAs or genomic DNAs from organisms other than the one from which thenucleic acid was isolated. For example, the other organisms may berelated organisms. In such procedures, a nucleic acid sample iscontacted with the probe under conditions which permit the probe tospecifically hybridize to related sequences. Hybridization of the probeto nucleic acids from the related organism is then detected using any ofthe methods described above.

By varying the stringency of the hybridization conditions used toidentify nucleic acids, such as cDNAs or genomic DNAs, which hybridizeto the detectable probe, nucleic acids having different levels ofhomology to the probe can be identified and isolated. Stringency may bevaried by conducting the hybridization at varying temperatures below themelting temperatures of the probes. The melting temperature, T_(m), isthe temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly complementary probe. Verystringent conditions are selected to be equal to or about 5° C. lowerthan the T_(m) for a particular probe. The melting temperature of theprobe may be calculated using the following formulas:

For probes between 14 and 70 nucleotides in length the meltingtemperature (T_(m)) is calculated using the formula: T_(m)=81.5+16.6(log[Na+])+0.41(fraction G+C)−(600/N) where N is the length of the probe.

If the hybridization is carried out in a solution containing formamide,the melting temperature may be calculated using the equation:T_(m)=81.5+16.6(log [Na+])+0.41(fraction G+C)−(0.63% formamide)−(600/N)where N is the length of the probe.

Prehybridization may be carried out in 6×SSC, 5×Denhardt's reagent, 0.5%SDS, 100 μg/ml denatured fragmented salmon sperm DNA or 6×SSC,5×Denhardt's reagent, 0.5% SDS, 100 μg/ml denatured fragmented salmonsperm DNA, 50% formamide. The formulas for SSC and Denhardt's solutionsare listed in Sambrook et al., supra.

In some embodiments, hybridization is conducted by adding the detectableprobe to the prehybridization solutions listed above. Where the probecomprises double stranded DNA, it is denatured before addition to thehybridization solution. In some embodiments, the filter is contactedwith the hybridization solution for a sufficient period of time to allowthe probe to hybridize to cDNAs or genomic DNAs containing sequencescomplementary thereto or homologous thereto. For probes over 200nucleotides in length, the hybridization may be carried out at 15-25° C.below the T_(m). For shorter probes, such as oligonucleotide probes, thehybridization may be conducted at 5-10° C. below the T_(m). In someembodiments, for hybridizations in 6×SSC, the hybridization is conductedat approximately 68° C. Usually, for hybridizations in 50% formamidecontaining solutions, the hybridization is conducted at approximately42° C.

Inhibiting Expression of Aldolase Enzymes

The invention provides nucleic acids complementary to (such as antisensesequences to) the nucleic acids in accordance with the invention, suchas aldolase enzyme-encoding nucleic acids, such as nucleic acidscomprising antisense, siRNA, miRNA, ribozymes. Nucleic acids inaccordance with the invention comprising antisense sequences can becapable of inhibiting the transport, splicing or transcription ofaldolase enzyme-encoding genes. The inhibition can be effected throughthe targeting of genomic DNA or messenger RNA. The transcription orfunction of targeted nucleic acid can be inhibited, for example, byhybridization and/or cleavage. One exemplary set of inhibitors providedby the present invention includes oligonucleotides which are able toeither bind aldolase, such as pyruvate aldolase, such as HMG and/or KHGaldolase enzyme gene or message, in either case preventing or inhibitingthe production or function of an aldolase, such as pyruvate aldolase,HMG and/or KHG aldolase enzyme. The association can be through sequencespecific hybridization. Another useful class of inhibitors includesoligonucleotides which cause inactivation or cleavage of aldolase, suchas pyruvate aldolase, such as HMG and/or KHG aldolase enzyme message.The oligonucleotide can have enzyme activity which causes such cleavage,such as ribozymes. The oligonucleotide can be chemically modified orconjugated to an enzyme or composition capable of cleaving thecomplementary nucleic acid. A pool of many different sucholigonucleotides can be screened for those with the desired activity.Thus, the invention provides various compositions for the inhibition ofaldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme expression on a nucleic acid and/or protein level, such asantisense, siRNA, miRNA and ribozymes comprising aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzyme sequences inaccordance with the invention and the anti-aldolase, such asanti-pyruvate aldolase, such as anti-HMG and/or anti-KHG aldolaseantibodies in accordance with the invention.

Inhibition of aldolase, such as pyruvate aldolase, such as HMG and/orKHG aldolase enzyme expression can have a variety of industrialapplications. For example, inhibition of aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzyme expression can slow orprevent spoilage. In some embodiments, use of compositions in accordancewith the invention that inhibit the expression and/or activity ofaldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzymes, such as antibodies, antisense oligonucleotides, ribozymes,siRNA and miRNA are used to slow or prevent spoilage. Thus, in someembodiments, the invention provides methods and compositions comprisingapplication onto a plant or plant product (such as a cereal, a grain, afruit, seed, root, leaf, etc.) antibodies, antisense oligonucleotides,ribozymes, siRNA and miRNA in accordance with the invention to slow orprevent spoilage. These compositions also can be expressed by the plant(such as a transgenic plant) or another organism (such as a bacterium orother microorganism transformed with an aldolase, such as pyruvatealdolase, HMG and/or KHG aldolase enzyme gene in accordance with theinvention).

The compositions in accordance with the invention for the inhibition ofaldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme expression (such as antisense, iRNA, ribozymes, antibodies) canbe used as pharmaceutical compositions, such as anti-pathogen agents orin other therapies, such as anti-microbials for, such as Salmonella.

Antisense Oligonucleotides

The invention provides antisense oligonucleotides capable of bindingaldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme message which, In some embodiments, can inhibit aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzyme, activity bytargeting mRNA. Strategies for designing antisense oligonucleotides arewell described in the scientific and patent literature, and the skilledartisan can design such aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzyme oligonucleotides using the novel reagents inaccordance with the invention. For example, gene walking/RNA mappingprotocols to screen for effective antisense oligonucleotides are wellknown in the art, see Ho (2000) Methods Enzymol. 314:168-183, describingan RNA mapping assay, which is based on standard molecular techniques toprovide an easy and reliable method for potent antisense sequenceselection. See also Smith (2000) Eur. J. Pharm. Sci. 11:191-198.

Naturally occurring nucleic acids are used as antisenseoligonucleotides. The antisense oligonucleotides can be of any length;for example, in other embodiments, the antisense oligonucleotides areabout 5 to about 100, about 10 to about 80, about 15 to about 60, about18 to about 40. The optimal length can be determined by routinescreening. The antisense oligonucleotides can be present at anyconcentration. The optimal concentration can be determined by routinescreening. A wide variety of synthetic, non-naturally occurringnucleotide and nucleic acid analogues are known which can address thispotential problem. For example, peptide nucleic acids (PNAs) containingnon-ionic backbones, such as N-(2-aminoethyl) glycine units can be used.Antisense oligonucleotides having phosphorothioate linkages can also beused, as described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol ApplPharmacol 144:189-197; Antisense Therapeutics, ed. Agrawal (HumanaPress, Totowa, N.J., 1996). Antisense oligonucleotides having syntheticDNA backbone analogues provided by the invention can also includephosphoro-dithioate, methylphosphonate, phosphoramidate, alkylphosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino),3′-N-carbamate, and morpholino carbamate nucleic acids, as describedabove.

Combinatorial chemistry methodology can be used to create vast numbersof oligonucleotides that can be rapidly screened for specificoligonucleotides that have appropriate binding affinities andspecificities toward any target, such as the sense and antisensealdolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme sequences in accordance with the invention (see Gold (1995) J. ofBiol. Chem. 270:13581-13584).

Inhibitory Ribozymes

The invention provides ribozymes capable of binding aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzyme message. Theseribozymes can inhibit aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzyme activity by, such as targeting mRNA.Strategies for designing ribozymes and selecting the aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzyme-specificantisense sequence for targeting are well described in the scientificand patent literature, and the skilled artisan can design such ribozymesusing the novel reagents in accordance with the invention. Ribozymes actby binding to a target RNA through the target RNA binding portion of aribozyme which is held in close proximity to an enzymatic portion of theRNA that cleaves the target RNA. Thus, the ribozyme recognizes and bindsa target RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cleave and inactivate the targetRNA. Cleavage of a target RNA in such a manner will destroy its abilityto direct synthesis of an encoded protein if the cleavage occurs in thecoding sequence. After a ribozyme has bound and cleaved its RNA target,it can be released from that RNA to bind and cleave new targetsrepeatedly.

In some circumstances, the enzymatic nature of a ribozyme can beadvantageous over other technologies, such as antisense technology(where a nucleic acid molecule simply binds to a nucleic acid target toblock its transcription, translation or association with anothermolecule) as the effective concentration of ribozyme necessary to effecta therapeutic treatment can be lower than that of an antisenseoligonucleotide. This potential advantage reflects the ability of theribozyme to act enzymatically. Thus, a single ribozyme molecule is ableto cleave many molecules of target RNA. In some embodiments, a ribozymeis a highly specific inhibitor, with the specificity of inhibitiondepending not only on the base pairing mechanism of binding, but also onthe mechanism by which the molecule inhibits the expression of the RNAto which it binds. That is, the inhibition is caused by cleavage of theRNA target and so specificity is defined as the ratio of the rate ofcleavage of the targeted RNA over the rate of cleavage of non-targetedRNA. This cleavage mechanism is dependent upon factors additional tothose involved in base pairing. Thus, the specificity of action of aribozyme can be greater than that of antisense oligonucleotide bindingthe same RNA site.

The ribozyme in accordance with the invention, such as an enzymaticribozyme RNA molecule, can be formed in a hammerhead motif, a hairpinmotif, as a hepatitis delta virus motif, a group I intron motif and/oran RNaseP-like RNA in association with an RNA guide sequence. Examplesof hammerhead motifs are described by, such as Rossi (1992) AidsResearch and Human Retroviruses 8:183; hairpin motifs by Hampel (1989)Biochemistry 28:4929, and Hampel (1990) Nuc. Acids Res. 18:299; thehepatitis delta virus motif by Perrotta (1992) Biochemistry 31:16; theRNaseP motif by Guerrier-Takada (1983) Cell 35:849; and the group Iintron by Cech U.S. Pat. No. 4,987,071. The recitation of these specificmotifs is not intended to be limiting. Those skilled in the art willrecognize that a ribozyme in accordance with the invention, such as anenzymatic RNA molecule of this invention, can have a specific substratebinding site complementary to one or more of the target gene RNAregions. A ribozyme in accordance with the invention can have anucleotide sequence within or surrounding that substrate binding sitewhich imparts an RNA cleaving activity to the molecule.

RNA Interference (RNAi)

In some embodiments, the invention provides RNA inhibitory molecules,so-called “RNAi” molecules, comprising aldolase, such as pyruvatealdolase, HMG and/or KHG aldolase enzyme sequences in accordance withthe invention. The RNAi molecule can comprise a double-stranded RNA(dsRNA) molecule, such as siRNA, miRNA and/or short hairpin RNA (shRNA)molecules. The RNAi molecule, such as siRNA (small inhibitory RNA)and/or miRNA (micro RNA), can inhibit expression of an aldolase, such aspyruvate aldolase, HMG and/or KHG aldolase enzyme gene. In someembodiments, the RNAi molecule, such as siRNA and/or miRNA, is about 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29or more duplex nucleotides in length. While the invention is not limitedby any particular mechanism of action, the RNAi can enter a cell andcause the degradation of a single-stranded RNA (ssRNA) of similar oridentical sequences, including endogenous mRNAs. When a cell is exposedto double-stranded RNA (dsRNA), mRNA from the homologous gene isselectively degraded by a process called RNA interference (RNAi). Apossible basic mechanism behind RNAi is the breaking of adouble-stranded RNA (dsRNA) matching a specific gene sequence into shortpieces called short interfering RNA, which trigger the degradation ofmRNA that matches its sequence. In some embodiments, the RNAi's inaccordance with the invention are used in gene-silencing therapeutics,see Shuey (2002) Drug Discov. Today 7:1040-1046. In some embodiments,the invention provides methods to selectively degrade RNA using theRNAi's molecules, such as siRNA and/or miRNA, in accordance with theinvention. The process may be practiced in vitro, ex vivo or in vivo. Insome embodiments, the RNAi molecules in accordance with the inventioncan be used to generate a loss-of-function mutation in a cell, an organor an animal.

In one aspect, intracellular introduction of the RNAi is byinternalization of a target cell specific ligand bonded to an RNAbinding protein comprising an RNAi (such as microRNA) is adsorbed. Theligand is specific to a unique target cell surface antigen. The ligandcan be spontaneously internalized after binding to the cell surfaceantigen. If the unique cell surface antigen is not naturallyinternalized after binding to its ligand, internalization can bepromoted by the incorporation of an arginine-rich peptide, or othermembrane permeable peptide, into the structure of the ligand or RNAbinding protein or attachment of such a peptide to the ligand or RNAbinding protein. See U.S. Patent App. Pub. Nos. 20060030003;20060025361; 20060019286; 20060019258. In one aspect, the inventionprovides lipid-based formulations for delivering, such as introducingnucleic acids of the invention as nucleic acid-lipid particlescomprising an RNAi molecule to a cell, see .g., U.S. Patent App. Pub.No. 20060008910.

Modification of Nucleic Acids—Making Variant Enzymes of the Invention

The invention provides methods of generating variants of the nucleicacids in accordance with the invention, such as those encoding analdolase, such as pyruvate aldolase, HMG and/or KHG aldolase enzyme.These methods can be repeated or used in various combinations togenerate aldolase, such as pyruvate aldolase, such as HMG and/or KHGaldolase enzymes having an altered or different activity or an alteredor different stability from that of an aldolase, such as pyruvatealdolase, HMG and/or KHG aldolase enzyme encoded by the template nucleicacid. These methods also can be repeated or used in variouscombinations, such as to generate variations in gene/message expression,message translation or message stability. In other embodiments, thegenetic composition of a cell is altered by, such as modification of ahomologous gene ex vivo, followed by its reinsertion into the cell.

A nucleic acid in accordance with the invention can be altered by anymeans. For example, random or stochastic methods, or, non-stochastic, or“directed evolution,” methods, see U.S. Pat. No. 6,361,974. Methods forrandom mutation of genes are well known in the art, see U.S. Pat. No.5,830,696. For example, mutagens can be used to randomly mutate a gene.Mutagens include, such as ultraviolet light or gamma irradiation, or achemical mutagen, such as mitomycin, nitrous acid, photoactivatedpsoralens, alone or in combination, to induce DNA breaks amenable torepair by recombination. Other chemical mutagens include, for example,sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.Other mutagens are analogues of nucleotide precursors, such asnitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Theseagents can be added to a PCR reaction in place of the nucleotideprecursor thereby mutating the sequence. Intercalating agents such asproflavine, acriflavine, quinacrine and the like can also be used.

Any technique in molecular biology can be used, such as random PCRmutagenesis, see Rice (1992) Proc. Natl. Acad. Sci. USA 89:5467-5471;or, combinatorial multiple cassette mutagenesis, see Crameri (1995)Biotechniques 18:194-196. Alternatively, nucleic acids, such as genes,can be reassembled after random, or “stochastic,” fragmentation, seeU.S. Pat. Nos. 6,291,242; 6,287,862; 6,287,861; 5,955,358; 5,830,721;5,824,514; 5,811,238; 5,605,793. In other embodiments, modifications,additions or deletions are introduced by error-prone PCR, shuffling,oligonucleotide-directed mutagenesis, assembly PCR, sexual PCRmutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly (such as GeneReassembly, see U.S. Pat. No.6,537,776), Gene Site Saturation Mutagenesis (GSSM), synthetic ligationreassembly (SLR), recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation, Chromosomal Saturation Mutagenesis (CSM) and/or acombination of these and other methods.

The following publications describe a variety of recursive recombinationprocedures and/or methods which can be incorporated into the methods inaccordance with the invention: Stemmer (1999) “Molecular breeding ofviruses for targeting and other clinical properties” Tumor Targeting4:1-4; Ness (1999) Nature Biotechnology 17:893-896; Chang (1999)“Evolution of a cytokine using DNA family shuffling” NatureBiotechnology 17:793-797; Minshull (1999) “Protein evolution bymolecular breeding” Current Opinion in Chemical Biology 3:284-290;Christians (1999) “Directed evolution of thymidine kinase for AZTphosphorylation using DNA family shuffling” Nature Biotechnology17:259-264; Crameri (1998) “DNA shuffling of a family of genes fromdiverse species accelerates directed evolution” Nature 391:288-291;Crameri (1997) “Molecular evolution of an arsenate detoxificationpathway by DNA shuffling,” Nature Biotechnology 15:436-438; Zhang (1997)“Directed evolution of an effective fucosidase from a galactosidase byDNA shuffling and screening” Proc. Natl. Acad. Sci. USA 94:4504-4509;Patten et al. (1997) “applications of DNA Shuffling to Pharmaceuticalsand Vaccines” Current Opinion in Biotechnology 8:724-733; Crameri et al.(1996) “Construction and evolution of antibody-phage libraries by DNAshuffling” Nature Medicine 2:100-103; Gates et al. (1996) “Affinityselective isolation of ligands from peptide libraries through display ona lac repressor ‘headpiece dimer’” Journal of Molecular Biology255:373-386; Stemmer (1996) “Sexual PCR and Assembly PCR” In: TheEncyclopedia of Molecular Biology. VCH Publishers, New York. pp.447-457; Crameri and Stemmer (1995) “Combinatorial multiple cassettemutagenesis creates all the permutations of mutant and wildtypecassettes” BioTechniques 18:194-195; Stemmer et al. (1995) “Single-stepassembly of a gene and entire plasmid form large numbers ofoligodeoxyribonucleotides” Gene, 164:49-53; Stemmer (1995) “TheEvolution of Molecular Computation” Science 270: 1510; Stemmer (1995)“Searching Sequence Space” Bio/Technology 13:549-553; Stemmer (1994)“Rapid evolution of a protein in vitro by DNA shuffling” Nature370:389-391; and Stemmer (1994) “DNA shuffling by random fragmentationand reassembly: In vitro recombination for molecular evolution.” Proc.Natl. Acad. Sci. USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al. (1997) “Approaches to DNAmutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al.(1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller (1987)Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Taylor(1985) “The use of phosphorothioate-modified DNA in restriction enzymereactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764; Taylor(1985) “The rapid generation of oligonucleotide-directed mutations athigh frequency using phosphorothioate-modified DNA” Nucl. Acids Res. 13:8765-8787 (1985); Nakamaye (1986) “Inhibition of restrictionendonuclease Nci I cleavage by phosphorothioate groups and itsapplication to oligonucleotide-directed mutagenesis” Nucl. Acids Res.14: 9679-9698; Sayers (1988) “Y-T Exonucleases in phosphorothioate-basedoligonucleotide-directed mutagenesis” Nucl. Acids Res. 16:791-802; andSayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350-367; Kramer (1988) “Improved enzymatic invitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999).

Additional protocols that can be used to practice the invention includepoint mismatch repair (Kramer (1984) “Point Mismatch Repair” Cell38:879-887), mutagenesis using repair-deficient host strains (Carter etal. (1985) “Improved oligonucleotide site-directed mutagenesis using M13vectors” Nucl. Acids Res. 13: 4431-4443; and Carter (1987) “Improvedoligonucleotide-directed mutagenesis using M13 vectors” Methods inEnzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) “Useof oligonucleotides to generate large deletions” Nucl. Acids Res. 14:5115), restriction-selection and restriction-selection andrestriction-purification (Wells et al. (1986) “Importance ofhydrogen-bond formation in stabilizing the transition state ofsubtilisin” Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis bytotal gene synthesis (Nambiar et al. (1984) “Total synthesis and cloningof a gene coding for the ribonuclease S protein” Science 223: 1299-1301;Sakamar and Khorana (1988) “Total synthesis and expression of a gene forthe a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin)” Nucl. Acids Res. 14: 6361-6372; Wells et al.(1985) “Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites” Gene 34:315-323; and Grundstrom etal. (1985) “Oligonucleotide-directed mutagenesis by microscale‘shot-gun’ gene synthesis” Nucl. Acids Res. 13: 3305-3316),double-strand break repair (Mandecki (1986); Arnold (1993) “Proteinengineering for unusual environments” Current Opinion in Biotechnology4:450-455. “Oligonucleotide-directed double-strand break repair inplasmids of Escherichia coli: a method for site-specific mutagenesis”Proc. Natl. Acad. Sci. USA, 83:7177-7181). Additional details on many ofthe above methods can be found in Methods in Enzymology Volume 154,which also describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Protocols that can be used to practice the invention are described, suchas in U.S. Pat. Nos. 5,605,793 to Stemmer (Feb. 25, 1997), “Methods forIn Vitro Recombination;” U.S. Pat. No. 5,811,238 to Stemmer et al. (Sep.22, 1998) “Methods for Generating Polynucleotides having DesiredCharacteristics by Iterative Selection and Recombination;” U.S. Pat. No.5,830,721 to Stemmer et al. (Nov. 3, 1998), “DNA Mutagenesis by RandomFragmentation and Reassembly;” U.S. Pat. No. 5,834,252 to Stemmer, etal. (Nov. 10, 1998) “End-Complementary Polymerase Reaction;” U.S. Pat.No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), “Methods andCompositions for Cellular and Metabolic Engineering;” WO 95/22625,Stemmer and Crameri, “Mutagenesis by Random Fragmentation andReassembly;” WO 96/33207 by Stemmer and Lipschutz “End ComplementaryPolymerase Chain Reaction;” WO 97/20078 by Stemmer and Crameri “Methodsfor Generating Polynucleotides having Desired Characteristics byIterative Selection and Recombination;” WO 97/35966 by Minshull andStemmer, “Methods and Compositions for Cellular and MetabolicEngineering;” WO 99/41402 by Punnonen et al. “Targeting of GeneticVaccine Vectors;” WO 99/41383 by Punnonen et al. “Antigen LibraryImmunization;” WO 99/41369 by Punnonen et al. “Genetic Vaccine VectorEngineering;” WO 99/41368 by Punnonen et al. “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” WO 98/27230 by Stemmer etal., “Methods for Optimization of Gene Therapy by Recursive SequenceShuffling and Selection,” WO 00/00632, “Methods for Generating HighlyDiverse Libraries,” WO 00/09679, “Methods for Obtaining in VitroRecombined Polynucleotide Sequence Banks and Resulting Sequences,” WO98/42832 by Arnold et al., “Recombination of Polynucleotide SequencesUsing Random or Defined Primers,” WO 99/29902 by Arnold et al., “Methodfor Creating Polynucleotide and Polypeptide Sequences,” WO 98/41653 byVind, “An in Vitro Method for Construction of a DNA Library,” WO98/41622 by Borchert et al., “Method for Constructing a Library UsingDNA Shuffling,” and WO 98/42727 by Pati and Zarling, “SequenceAlterations using Homologous Recombination.”

Protocols that can be used to practice the invention (providing detailsregarding various diversity generating methods) are described, such asin U.S. patent application Ser. No. 09/407,800, “SHUFFLING OF CODONALTERED GENES” by Patten et al. filed Sep. 28, 1999; “EVOLUTION OF WHOLECELLS AND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION” by del Cardayreet al., U.S. Pat. No. 6,379,964; “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACIDRECOMBINATION” by Crameri et al., U.S. Pat. Nos. 6,319,714; 6,368,861;6,376,246; 6,423,542; 6,426,224 and PCT/US00/01203; “USE OF CODON-VARIEDOLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” by Welch et al., U.S.Pat. No. 6,436,675; “METHODS FOR MAKING CHARACTER STRINGS,POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” bySelifonov et al., filed Jan. 18, 2000, (PCT/US00/01202) and, such as“METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDESHAVING DESIRED CHARACTERISTICS” by Selifonov et al., filed Jul. 18, 2000(U.S. Ser. No. 09/618,579); “METHODS OF POPULATING DATA STRUCTURES FORUSE IN EVOLUTIONARY SIMULATIONS” by Selifonov and Stemmer, filed Jan.18, 2000 (PCT/US00/01138); and “SINGLE-STRANDED NUCLEIC ACIDTEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION” byAffholter, filed Sep. 6, 2000 (U.S. Ser. No. 09/656,549); and U.S. Pat.Nos. 6,177,263; 6,153,410.

Non-stochastic, or “directed evolution,” methods include, such assaturation mutagenesis, such as Gene Site Saturation Mutagenesis (GSSM),synthetic ligation reassembly (SLR), or a combination thereof are usedto modify the nucleic acids in accordance with the invention to generatealdolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzymes with new or altered properties (such as activity under highlyacidic or alkaline conditions, high or low temperatures, and the like).Polypeptides encoded by the modified nucleic acids can be screened foran activity before testing for carbon-carbon bond formation or cleavageor other activity. Any testing modality or protocol can be used, such asusing a capillary array platform. See U.S. Pat. Nos. 6,361,974;6,280,926; 5,939,250.

Gene Site Saturation Mutagenesis, or, GSSM

The invention also provides methods for making enzyme using Gene SiteSaturation mutagenesis, or, GSSM, as described herein, and also in U.S.Pat. Nos. 6,171,820 and 6,579,258. In some embodiments, codon primerscontaining a degenerate N,N,G/T sequence are used to introduce pointmutations into a polynucleotide, such as an aldolase, such as pyruvatealdolase, HMG and/or KHG aldolase enzyme or an antibody in accordancewith the invention, so as to generate a set of progeny polypeptides inwhich a full range of single amino acid substitutions is represented ateach amino acid position, such as an amino acid residue in an enzymeactive site or ligand binding site targeted to be modified. Theseoligonucleotides can comprise a contiguous first homologous sequence, adegenerate N,N,G/T sequence, and, optionally, a second homologoussequence. The downstream progeny translational products from the use ofsuch oligonucleotides include all possible amino acid changes at eachamino acid site along the polypeptide, because the degeneracy of theN,N,G/T sequence includes codons for all 20 amino acids. In someembodiments, one such degenerate oligonucleotide (comprised of, such asone degenerate N,N,G/T cassette) is used for subjecting each originalcodon in a parental polynucleotide template to a full range of codonsubstitutions. In other embodiments, at least two degenerate cassettesare used—either in the same oligonucleotide or not, for subjecting atleast two original codons in a parental polynucleotide template to afull range of codon substitutions. For example, more than one N,N,G/Tsequence can be contained in one oligonucleotide to introduce amino acidmutations at more than one site. This plurality of N,N,G/T sequences canbe directly contiguous, or separated by one or more additionalnucleotide sequence(s). In other embodiments, oligonucleotidesserviceable for introducing additions and deletions can be used eitheralone or in combination with the codons containing an N,N,G/T sequence,to introduce any combination or permutation of amino acid additions,deletions, and/or substitutions.

In some embodiments, simultaneous mutagenesis of two or more contiguousamino acid positions is done using an oligonucleotide that containscontiguous N,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence. Inother embodiments, degenerate cassettes having less degeneracy than theN,N,G/T sequence are used. For example, it may be desirable in someinstances to use (such as in an oligonucleotide) a degenerate tripletsequence comprised of only one N, where said N can be in the firstsecond or third position of the triplet. Any other bases including anycombinations and permutations thereof can be used in the remaining twopositions of the triplet. Alternatively, it may be desirable in someinstances to use (such as in an oligo) a degenerate N,N,N tripletsequence.

In some embodiments, use of degenerate triplets (such as N,N,G/Ttriplets) allows for systematic and easy generation of a full range ofpossible natural amino acids (for a total of 20 amino acids) into eachand every amino acid position in a polypeptide (in other embodiments,the methods also include generation of less than all possiblesubstitutions per amino acid residue, or codon, position). For example,for a 100 amino acid polypeptide, 2000 distinct species (i.e. 20possible amino acids per position X 100 amino acid positions) can begenerated. Through the use of an oligonucleotide or set ofoligonucleotides containing a degenerate N,N,G/T triplet, 32 individualsequences can code for all 20 possible natural amino acids. Thus, in areaction vessel in which a parental polynucleotide sequence is subjectedto saturation mutagenesis using at least one such oligonucleotide, thereare generated 32 distinct progeny polynucleotides encoding 20 distinctpolypeptides. In contrast, the use of a non-degenerate oligonucleotidein site-directed mutagenesis leads to only one progeny polypeptideproduct per reaction vessel. Nondegenerate oligonucleotides canoptionally be used in combination with degenerate primers disclosed; forexample, nondegenerate oligonucleotides can be used to generate specificpoint mutations in a working polynucleotide. This provides one means togenerate specific silent point mutations, point mutations leading tocorresponding amino acid changes, and point mutations that cause thegeneration of stop codons and the corresponding expression ofpolypeptide fragments.

In some embodiments, each saturation mutagenesis reaction vesselcontains polynucleotides encoding at least 20 progeny polypeptide (suchas aldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzymes) molecules such that all 20 natural amino acids are representedat the one specific amino acid position corresponding to the codonposition mutagenized in the parental polynucleotide (other embodimentsuse less than all 20 natural combinations). The 32-fold degenerateprogeny polypeptides generated from each saturation mutagenesis reactionvessel can be subjected to clonal amplification (such as cloned into asuitable host, such as E. coli host, using, such as an expressionvector) and subjected to expression screening. When an individualprogeny polypeptide is identified by screening to display a favorablechange in property (when compared to the parental polypeptide, such asincreased carbon-carbon formation or cleavage activity under alkaline oracidic conditions), it can be sequenced to identify the correspondinglyfavorable amino acid substitution contained therein.

In some embodiments, upon mutagenizing each and every amino acidposition in a parental polypeptide using saturation mutagenesis asdisclosed herein, favorable amino acid changes may be identified at morethan one amino acid position. One or more new progeny molecules can begenerated that contain a combination of all or part of these favorableamino acid substitutions. For example, if 2 specific favorable aminoacid changes are identified in each of 3 amino acid positions in apolypeptide, the permutations include 3 possibilities at each position(no change from the original amino acid, and each of two favorablechanges) and 3 positions. Thus, there are 3×3×3 or 27 totalpossibilities, including 7 that were previously examined—6 single pointmutations (i.e. 2 at each of three positions) and no change at anyposition.

In yet another embodiment, site-saturation mutagenesis can be usedtogether with shuffling, chimerization, recombination and othermutagenizing processes, along with screening. This invention providesfor the use of any mutagenizing process(es), including saturationmutagenesis, in an iterative manner. In one exemplification, theiterative use of any mutagenizing process(es) is used in combinationwith screening.

The invention also provides for the use of proprietary codon primers(containing a degenerate N,N,N sequence) to introduce point mutationsinto a polynucleotide, so as to generate a set of progeny polypeptidesin which a full range of single amino acid substitutions is representedat each amino acid position (Gene Site Saturation Mutagenesis (GSSM)).The oligos used are comprised contiguously of a first homologoussequence, a degenerate N,N,N sequence and, in some embodiments but notnecessarily, a second homologous sequence. The downstream progenytranslational products from the use of such oligos include all possibleamino acid changes at each amino acid site along the polypeptide,because the degeneracy of the N,N,N sequence includes codons for all 20amino acids.

In some embodiments, one such degenerate oligo (comprised of onedegenerate N,N,N cassette) is used for subjecting each original codon ina parental polynucleotide template to a full range of codonsubstitutions. In other embodiments, at least two degenerate N,N,Ncassettes are used—either in the same oligo or not, for subjecting atleast two original codons in a parental polynucleotide template to afull range of codon substitutions. Thus, more than one N,N,N sequencecan be contained in one oligo to introduce amino acid mutations at morethan one site. This plurality of N,N,N sequences can be directlycontiguous, or separated by one or more additional nucleotidesequence(s). In other embodiments, oligos serviceable for introducingadditions and deletions can be used either alone or in combination withthe codons containing an N,N,N sequence, to introduce any combination orpermutation of amino acid additions, deletions and/or substitutions.

In some embodiments, it is possible to simultaneously mutagenize two ormore contiguous amino acid positions using an oligo that containscontiguous N,N,N triplets, i.e. a degenerate (N,N,N)_(n) sequence. Inother embodiments, the present invention provides for the use ofdegenerate cassettes having less degeneracy than the N,N,N sequence. Forexample, it may be desirable in some instances to use (such as in anoligo) a degenerate triplet sequence comprised of only one N, where theN can be in the first second or third position of the triplet. Any otherbases including any combinations and permutations thereof can be used inthe remaining two positions of the triplet. Alternatively, it may bedesirable in some instances to use (such as in an oligo) a degenerateN,N,N triplet sequence, N,N,G/T, or an N,N, G/C triplet sequence.

In some embodiments, use of a degenerate triplet (such as N,N,G/T or anN,N, G/C triplet sequence) is advantageous for several reasons. In someembodiments, this invention provides means to systematically and fairlyeasily generate the substitution of the full range of possible aminoacids (for a total of 20 amino acids) into each and every amino acidposition in a polypeptide. Thus, for a 100 amino acid polypeptide, theinvention provides ways to systematically and fairly easily generate2000 distinct species (i.e., 20 possible amino acids per position times100 amino acid positions). It is appreciated that there is provided,through the use of an oligo containing a degenerate N,N,G/T or an N,N,G/C triplet sequence, 32 individual sequences that code for 20 possibleamino acids. Thus, in a reaction vessel in which a parentalpolynucleotide sequence is subjected to saturation mutagenesis using onesuch oligo, there are generated 32 distinct progeny polynucleotidesencoding 20 distinct polypeptides. In contrast, the use of anon-degenerate oligo in site-directed mutagenesis leads to only oneprogeny polypeptide product per reaction vessel.

This invention also provides for the use of nondegenerate oligos, whichcan optionally be used in combination with degenerate primers disclosed.It is appreciated that in some situations, it is advantageous to usenondegenerate oligos to generate specific point mutations in a workingpolynucleotide. This provides means to generate specific silent pointmutations, point mutations leading to corresponding amino acid changesand point mutations that cause the generation of stop codons and thecorresponding expression of polypeptide fragments.

Thus, in some embodiments of this invention, each saturation mutagenesisreaction vessel contains polynucleotides encoding at least 20 progenypolypeptide molecules such that all 20 amino acids are represented atthe one specific amino acid position corresponding to the codon positionmutagenized in the parental polynucleotide. The 32-fold degenerateprogeny polypeptides generated from each saturation mutagenesis reactionvessel can be subjected to clonal amplification (such as cloned into asuitable E. coli host using an expression vector) and subjected toexpression screening. When an individual progeny polypeptide isidentified by screening to display a favorable change in property (whencompared to the parental polypeptide), it can be sequenced to identifythe correspondingly favorable amino acid substitution contained therein.

In some embodiments, upon mutagenizing each and every amino acidposition in a parental polypeptide using saturation mutagenesis asdisclosed herein, a favorable amino acid changes is identified at morethan one amino acid position. One or more new progeny molecules can begenerated that contain a combination of all or part of these favorableamino acid substitutions. For example, if 2 specific favorable aminoacid changes are identified in each of 3 amino acid positions in apolypeptide, the permutations include 3 possibilities at each position(no change from the original amino acid and each of two favorablechanges) and 3 positions. Thus, there are 3×3×3 or 27 totalpossibilities, including 7 that were previously examined—6 single pointmutations (i.e., 2 at each of three positions) and no change at anyposition.

The invention provides for the use of saturation mutagenesis incombination with additional mutagenization processes, such as processwhere two or more related polynucleotides are introduced into a suitablehost cell such that a hybrid polynucleotide is generated byrecombination and reductive reassortment.

In addition to performing mutagenesis along the entire sequence of agene, the instant invention provides that mutagenesis can be use toreplace each of any number of bases in a polynucleotide sequence,wherein the number of bases to be mutagenized is, in some embodimentsevery integer from 15 to 100,000. Thus, instead of mutagenizing everyposition along a molecule, one can subject every or a discrete number ofbases (in some embodiments a subset totaling from 15 to 100,000) tomutagenesis. In some embodiments, a separate nucleotide is used formutagenizing each position or group of positions along a polynucleotidesequence. A group of 3 positions to be mutagenized may be a codon. Themutations can be introduced using a mutagenic primer, containing aheterologous cassette, also referred to as a mutagenic cassette.Exemplary cassettes can have from 1 to 500 bases. Each nucleotideposition in such heterologous cassettes be N, A, C, G, T, A/C, A/G, A/T,C/G, C/T, G/T, C/G/T, A/G/T, A/C/T, A/C/G, or E, where E is any basethat is not A, C, G, or T (E can be referred to as a designer oligo).

In some embodiments, saturation mutagenesis is comprised of mutagenizinga complete set of mutagenic cassettes (wherein each cassette is, in someembodiments, about 1-500 bases in length) in defined polynucleotidesequence to be mutagenized (wherein the sequence to be mutagenized is,in some embodiments, from about 15 to 100,000 bases in length). Thus, agroup of mutations (ranging from 1 to 100 mutations) is introduced intoeach cassette to be mutagenized. A grouping of mutations to beintroduced into one cassette can be different or the same from a secondgrouping of mutations to be introduced into a second cassette during theapplication of one round of saturation mutagenesis. Such groupings areexemplified by deletions, additions, groupings of particular codons andgroupings of particular nucleotide cassettes.

In some embodiments, defined sequences to be mutagenized include a wholegene, pathway, cDNA, an entire open reading frame (ORF) and entirepromoter, enhancer, repressor/transactivator, origin of replication,intron, operator, or any polynucleotide functional group. Generally, a“defined sequences” for this purpose may be any polynucleotide that a 15base-polynucleotide sequence and polynucleotide sequences of lengthsbetween 15 bases and 15,000 bases (this invention specifically namesevery integer in between). Considerations in choosing groupings ofcodons include types of amino acids encoded by a degenerate mutageniccassette.

In some embodiments, a grouping of mutations that can be introduced intoa mutagenic cassette, this invention specifically provides fordegenerate codon substitutions (using degenerate oligos) that code for2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20amino acids at each position and a library of polypeptides encodedthereby.

Synthetic Ligation Reassembly (SLR)

The invention provides a non-stochastic gene modification system termed“synthetic ligation reassembly,” or simply “SLR,” a “directed evolutionprocess,” to generate polypeptides, such as aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzymes or antibodies inaccordance with the invention, with new or altered properties.

SLR is a method of ligating oligonucleotide fragments togethernon-stochastically. This method differs from stochastic oligonucleotideshuffling in that the nucleic acid building blocks are not shuffled,concatenated or chimerized randomly, but rather are assemblednon-stochastically. See U.S. Pat. Nos. 6,773,900; 6,740,506; 6,713,282;6,635,449; 6,605,449; 6,537,776. In some embodiments, SLR comprises thefollowing steps: (a) providing a template polynucleotide, wherein thetemplate polynucleotide comprises sequence encoding a homologous gene;(b) providing a plurality of building block polynucleotides, wherein thebuilding block polynucleotides are designed to cross-over reassemblewith the template polynucleotide at a predetermined sequence, and abuilding block polynucleotide comprises a sequence that is a variant ofthe homologous gene and a sequence homologous to the templatepolynucleotide flanking the variant sequence; (c) combining a buildingblock polynucleotide with a template polynucleotide such that thebuilding block polynucleotide cross-over reassembles with the templatepolynucleotide to generate polynucleotides comprising homologous genesequence variations.

SLR does not depend on the presence of high levels of homology betweenpolynucleotides to be rearranged. Thus, this method can be used tonon-stochastically generate libraries (or sets) of progeny moleculescomprised of over 10¹⁰⁰ different chimeras. SLR can be used to generatelibraries comprised of over 10¹⁰⁰⁰ different progeny chimeras. Thus,embodiments of the present invention include non-stochastic methods ofproducing a set of finalized chimeric nucleic acid molecule shaving anoverall assembly order that is chosen by design. This method includesthe steps of generating by design a plurality of specific nucleic acidbuilding blocks having serviceable mutually compatible ligatable ends,and assembling these nucleic acid building blocks, such that a designedoverall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus, the overall assembly order in which thenucleic acid building blocks can be coupled is specified by the designof the ligatable ends. If more than one assembly step is to be used,then the overall assembly order in which the nucleic acid buildingblocks can be coupled is also specified by the sequential order of theassembly step(s). In some embodiments, the annealed building pieces aretreated with an enzyme, such as a ligase (such as T4 DNA ligase), toachieve covalent bonding of the building pieces.

In some embodiments, the design of the oligonucleotide building blocksis obtained by analyzing a set of progenitor nucleic acid sequencetemplates that serve as a basis for producing a progeny set of finalizedchimeric polynucleotides. These parental oligonucleotide templates thusserve as a source of sequence information that aids in the design of thenucleic acid building blocks that are to be mutagenized, such aschimerized or shuffled. In some embodiments of this method, thesequences of a plurality of parental nucleic acid templates are alignedin order to select one or more demarcation points. The demarcationpoints can be located at an area of homology, and are comprised of oneor more nucleotides. These demarcation points are, in some embodiments,shared by at least two of the progenitor templates. The demarcationpoints can thereby be used to delineate the boundaries ofoligonucleotide building blocks to be generated in order to rearrangethe parental polynucleotides. The demarcation points identified andselected in the progenitor molecules serve as potential chimerizationpoints in the assembly of the final chimeric progeny molecules. Ademarcation point can be an area of homology (comprised of at least onehomologous nucleotide base) shared by at least two parentalpolynucleotide sequences. Alternatively, a demarcation point can be anarea of homology that is shared by at least half of the parentalpolynucleotide sequences, or, it can be an area of homology that isshared by at least two thirds of the parental polynucleotide sequences.Even more, in some embodiments, a serviceable demarcation points is anarea of homology that is shared by at least three fourths of theparental polynucleotide sequences, or, it can be shared by at almost allof the parental polynucleotide sequences. In some embodiments, ademarcation point is an area of homology that is shared by all of theparental polynucleotide sequences.

In some embodiments, a ligation reassembly process is performedexhaustively in order to generate an exhaustive library of progenychimeric polynucleotides. In other words, all possible orderedcombinations of the nucleic acid building blocks are represented in theset of finalized chimeric nucleic acid molecules. At the same time, inother embodiments, the assembly order (i.e. the order of assembly ofeach building block in the 5′ to 3 sequence of each finalized chimericnucleic acid) in each combination is by design (or non-stochastic) asdescribed above. Because of the non-stochastic nature of this invention,the possibility of unwanted side products is greatly reduced.

In other embodiments, the ligation reassembly method is performedsystematically. For example, the method is performed in order togenerate a systematically compartmentalized library of progenymolecules, with compartments that can be screened systematically, suchas one by one. In other words this invention provides that, through theselective and judicious use of specific nucleic acid building blocks,coupled with the selective and judicious use of sequentially steppedassembly reactions, a design can be achieved where specific sets ofprogeny products are made in each of several reaction vessels. Thisallows a systematic examination and screening procedure to be performed.Thus, these methods allow a potentially very large number of progenymolecules to be examined systematically in smaller groups. Because ofits ability to perform chimerizations in a manner that is highlyflexible yet exhaustive and systematic as well, particularly when thereis a low level of homology among the progenitor molecules, these methodsprovide for the generation of a library (or set) comprised of a largenumber of progeny molecules. Because of the non-stochastic nature of theinstant ligation reassembly invention, the progeny molecules generatedin some embodiments comprise a library of finalized chimeric nucleicacid molecules having an overall assembly order that is chosen bydesign. The saturation mutagenesis and optimized directed evolutionmethods also can be used to generate different progeny molecularspecies. It is appreciated that the invention provides freedom of choiceand control regarding the selection of demarcation points, the size andnumber of the nucleic acid building blocks, and the size and design ofthe couplings. It is appreciated, furthermore, that the requirement forintermolecular homology is highly relaxed for the operability of thisinvention. In fact, demarcation points can even be chosen in areas oflittle or no intermolecular homology. For example, because of codonwobble, i.e. the degeneracy of codons, nucleotide substitutions can beintroduced into nucleic acid building blocks without altering the aminoacid originally encoded in the corresponding progenitor template.Alternatively, a codon can be altered such that the coding for anoriginally amino acid is altered. This invention provides that suchsubstitutions can be introduced into the nucleic acid building block inorder to increase the incidence of intermolecular homologous demarcationpoints and thus to allow an increased number of couplings to be achievedamong the building blocks, which in turn allows a greater number ofprogeny chimeric molecules to be generated.

Synthetic Gene Reassembly

In some embodiments, the present invention provides a non-stochasticmethod termed synthetic gene reassembly, that is somewhat related tostochastic shuffling, save that the nucleic acid building blocks are notshuffled or concatenated or chimerized randomly, but rather areassembled non-stochastically. See U.S. Pat. No. 6,537,776.

The synthetic gene reassembly method does not depend on the presence ofa high level of homology between polynucleotides to be shuffled. Theinvention can be used to non-stochastically generate libraries (or sets)of progeny molecules comprised of over 10¹⁰⁰ different chimeras.Conceivably, synthetic gene reassembly can even be used to generatelibraries comprised of over 10¹⁰⁰⁰ different progeny chimeras.

Thus, in some embodiments, the invention provides a non-stochasticmethod of producing a set of finalized chimeric nucleic acid moleculeshaving an overall assembly order that is chosen by design, which methodis comprised of the steps of generating by design a plurality ofspecific nucleic acid building blocks having serviceable mutuallycompatible ligatable ends and assembling these nucleic acid buildingblocks, such that a designed overall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus, in some embodiments, the overall assemblyorder in which the nucleic acid building blocks can be coupled isspecified by the design of the ligatable ends and, if more than oneassembly step is to be used, then the overall assembly order in whichthe nucleic acid building blocks can be coupled is also specified by thesequential order of the assembly step(s). In a one embodiment, of theinvention, the annealed building pieces are treated with an enzyme, suchas a ligase (such as T4 DNA ligase) to achieve covalent bonding of thebuilding pieces.

In a another embodiment, the design of nucleic acid building blocks isobtained upon analysis of the sequences of a set of progenitor nucleicacid templates that serve as a basis for producing a progeny set offinalized chimeric nucleic acid molecules. These progenitor nucleic acidtemplates thus serve as a source of sequence information that aids inthe design of the nucleic acid building blocks that are to bemutagenized, i.e. chimerized or shuffled.

In one exemplification, the invention provides for the chimerization ofa family of related genes and their encoded family of related products.In a particular exemplification, the encoded products are enzymes. Thealdolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzymes of the present invention can be mutagenized in accordance withthe methods described herein.

Thus according to one embodiment of the invention, the sequences of aplurality of progenitor nucleic acid templates (such as polynucleotidesin accordance with the invention) are aligned in order to select one ormore demarcation points, which demarcation points can be located at anarea of homology. The demarcation points can be used to delineate theboundaries of nucleic acid building blocks to be generated. Thus, thedemarcation points identified and selected in the progenitor moleculesserve as potential chimerization points in the assembly of the progenymolecules.

In some embodiments, a serviceable demarcation point is an area ofhomology (comprised of at least one homologous nucleotide base) sharedby at least two progenitor templates, but the demarcation point can bean area of homology that is shared by at least half of the progenitortemplates, at least two thirds of the progenitor templates, at leastthree fourths of the progenitor templates and in some embodiments atalmost all of the progenitor templates. Even more in some embodimentsstill a serviceable demarcation point is an area of homology that isshared by all of the progenitor templates.

In a one embodiment, the gene reassembly process is performedexhaustively in order to generate an exhaustive library. In other words,all possible ordered combinations of the nucleic acid building blocksare represented in the set of finalized chimeric nucleic acid molecules.At the same time, the assembly order (i.e. the order of assembly of eachbuilding block in the 5′ to 3 sequence of each finalized chimericnucleic acid) in each combination is by design (or non-stochastic).Because of the non-stochastic nature of the method, the possibility ofunwanted side products is greatly reduced.

In other embodiments, the method provides that the gene reassemblyprocess is performed systematically, for example to generate asystematically compartmentalized library, with compartments that can bescreened systematically, such as one by one. In other words theinvention provides that, through the selective and judicious use ofspecific nucleic acid building blocks, coupled with the selective andjudicious use of sequentially stepped assembly reactions, anexperimental design can be achieved where specific sets of progenyproducts are made in each of several reaction vessels. This allows asystematic examination and screening procedure to be performed. Thus, itallows a potentially very large number of progeny molecules to beexamined systematically in smaller groups.

Because of its ability to perform chimerizations in a manner that ishighly flexible yet exhaustive and systematic as well, particularly whenthere is a low level of homology among the progenitor molecules, theinstant invention provides for the generation of a library (or set)comprised of a large number of progeny molecules. Because of thenon-stochastic nature of the instant gene reassembly invention, theprogeny molecules generated in some embodiments comprise a library offinalized chimeric nucleic acid molecules having an overall assemblyorder that is chosen by design. In some embodiments, such a generatedlibrary is comprised of greater than 10³ to greater than 10¹⁰⁰⁰different progeny molecular species.

In some embodiments, a set of finalized chimeric nucleic acid molecules,produced as described is comprised of a polynucleotide encoding apolypeptide. In one embodiment, this polynucleotide is a gene, which maybe a man-made gene. In another embodiment, this polynucleotide is a genepathway, which may be a man-made gene pathway. In some embodiments, theinvention provides that one or more man-made genes generated by theinvention may be incorporated into a man-made gene pathway, such aspathway operable in a eukaryotic organism (including a plant).

In another exemplification, the synthetic nature of the step in whichthe building blocks are generated allows the design and introduction ofnucleotides (such as one or more nucleotides, which may be, for example,codons or introns or regulatory sequences) that can later be optionallyremoved in an in vitro process (such as by mutagenesis) or in an in vivoprocess (such as by utilizing the gene splicing ability of a hostorganism). It is appreciated that in many instances the introduction ofthese nucleotides may also be desirable for many other reasons inaddition to the potential benefit of creating a serviceable demarcationpoint.

Thus, in some embodiments, the invention provides that a nucleic acidbuilding block can be used to introduce an intron. Thus, the inventionprovides that functional introns may be introduced into a man-made genein accordance with the invention. In some embodiments, the inventionalso provides that functional introns may be introduced into a man-madegene pathway in accordance with the invention. Accordingly, theinvention provides for the generation of a chimeric polynucleotide thatis a man-made gene containing one (or more) artificially introducedintron(s).

The invention also provides for the generation of a chimericpolynucleotide that is a man-made gene pathway containing one (or more)artificially introduced intron(s). In some embodiments, the artificiallyintroduced intron(s) are functional in one or more host cells for genesplicing much in the way that naturally-occurring introns servefunctionally in gene splicing. In some embodiments, the inventionprovides processes of producing man-made intron-containingpolynucleotides to be introduced into host organisms for recombinationand/or splicing.

A man-made gene produced using the invention can also serve as asubstrate for recombination with another nucleic acid. Likewise, aman-made gene pathway produced using the invention can also serve as asubstrate for recombination with another nucleic acid. In someembodiments, the recombination is facilitated by, or occurs at, areas ofhomology between the man-made, intron-containing gene and a nucleicacid, which serves as a recombination partner. In some embodiments, therecombination partner may also be a nucleic acid generated by theinvention, including a man-made gene or a man-made gene pathway.Recombination may be facilitated by or may occur at areas of homologythat exist at the one (or more) artificially introduced intron(s) in theman-made gene.

In some embodiments, the synthetic gene reassembly method in accordancewith the invention utilizes a plurality of nucleic acid building blocks,each of which, in some embodiments, has two ligatable ends. The twoligatable ends on each nucleic acid building block may be two blunt ends(i.e. each having an overhang of zero nucleotides), or in someembodiments one blunt end and one overhang, or more in some embodimentsstill two overhangs. In some embodiments, a useful overhang for thispurpose may be a 3′ overhang or a 5′ overhang. Thus, a nucleic acidbuilding block may have a 3′ overhang or alternatively a 5′ overhang oralternatively two 3′ overhangs or alternatively two 5′ overhangs. Theoverall order in which the nucleic acid building blocks are assembled toform a finalized chimeric nucleic acid molecule is determined bypurposeful experimental design and is not random.

In some embodiments, a nucleic acid building block is generated bychemical synthesis of two single-stranded nucleic acids (also referredto as single-stranded oligos) and contacting them so as to allow them toanneal to form a double-stranded nucleic acid building block. Adouble-stranded nucleic acid building block can be of variable size. Thesizes of these building blocks can be small or large. Exemplary sizesfor building block range from 1 base pair (not including any overhangs)to 100,000 base pairs (not including any overhangs). Other exemplarysize ranges are also provided, which have lower limits of from 1 bp to10,000 bp (including every integer value in between) and upper limits offrom 2 bp to 100,000 bp (including every integer value in between).

Many methods exist by which a double-stranded nucleic acid buildingblock can be generated that is serviceable for the invention; and theseare known in the art and can be readily performed by the skilledartisan. In some embodiments, a double-stranded nucleic acid buildingblock is generated by first generating two single stranded nucleic acidsand allowing them to anneal to form a double-stranded nucleic acidbuilding block. The two strands of a double-stranded nucleic acidbuilding block may be complementary at every nucleotide apart from anythat form an overhang; thus containing no mismatches, apart from anyoverhang(s). In another embodiment, the two strands of a double-strandednucleic acid building block are complementary at fewer than everynucleotide apart from any that form an overhang. Thus, according to thisembodiment, a double-stranded nucleic acid building block can be used tointroduce codon degeneracy. In some embodiments the codon degeneracy isintroduced using the site-saturation mutagenesis described herein, usingone or more N,N,G/T cassettes or alternatively using one or more N,N,Ncassettes.

The in vivo recombination method in accordance with the invention can beperformed blindly on a pool of unknown hybrids or alleles of a specificpolynucleotide or sequence. However, it is not necessary to know theactual DNA or RNA sequence of the specific polynucleotide. The approachof using recombination within a mixed population of genes can be usefulfor the generation of any useful proteins, for example, an aldolase inaccordance with the invention or a variant thereof. This approach may beused to generate proteins having altered specificity or activity. Theapproach may also be useful for the generation of hybrid nucleic acidsequences, for example, promoter regions, introns, exons, enhancersequences, 31 untranslated regions or 51 untranslated regions of genes.Thus this approach may be used to generate genes having increased ratesof expression. This approach may also be useful in the study ofrepetitive DNA sequences. Finally, this approach may be useful to makeribozymes or aptamers in accordance with the invention.

In some embodiments the invention described herein is directed to theuse of repeated cycles of reductive reassortment, recombination andselection which allow for the directed molecular evolution of highlycomplex linear sequences, such as DNA, RNA or proteins thoroughrecombination.

Optimized Directed Evolution System

The invention provides a non-stochastic gene modification system termed“optimized directed evolution system” to generate polypeptides, such asaldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzymes or antibodies in accordance with the invention, with new oraltered properties. In some embodiments, optimized directed evolution isdirected to the use of repeated cycles of reductive reassortment,recombination and selection that allow for the directed molecularevolution of nucleic acids through recombination.

Optimized directed evolution allows generation of a large population ofevolved chimeric sequences, wherein the generated population issignificantly enriched for sequences that have a predetermined number ofcrossover events. A crossover event is a point in a chimeric sequencewhere a shift in sequence occurs from one parental variant to anotherparental variant. Such a point is normally at the juncture of whereoligonucleotides from two parents are ligated together to form a singlesequence. This method allows calculation of the correct concentrationsof oligonucleotide sequences so that the final chimeric population ofsequences is enriched for the chosen number of crossover events. Thisprovides more control over choosing chimeric variants having apredetermined number of crossover events.

In addition, this method provides convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. Previously, if one generated, for example, 10¹³ chimericmolecules during a reaction, it would be extremely difficult to testsuch a high number of chimeric variants for a particular activity.Moreover, a significant portion of the progeny population would have avery high number of crossover events which resulted in proteins thatwere less likely to have increased levels of a particular activity. Byusing these methods, the population of chimerics molecules can beenriched for those variants that have a particular number of crossoverevents. Thus, although one can still generate 10¹³ chimeric moleculesduring a reaction, each of the molecules chosen for further analysismost likely has, for example, only three crossover events. Because theresulting progeny population can be skewed to have a predeterminednumber of crossover events, the boundaries on the functional varietybetween the chimeric molecules is reduced. This provides a moremanageable number of variables when calculating which oligonucleotidefrom the original parental polynucleotides might be responsible foraffecting a particular trait.

One method for creating a chimeric progeny polynucleotide sequence is tocreate oligonucleotides corresponding to fragments or portions of eachparental sequence. Each oligonucleotide in some embodiments includes aunique region of overlap so that mixing the oligonucleotides togetherresults in a new variant that has each oligonucleotide fragmentassembled in the correct order. Alternatively protocols for practicingthese methods in accordance with the invention can be found in U.S. Pat.Nos. 6,773,900; 6,740,506; 6,713,282; 6,635,449; 6,605,449; 6,537,776;6,361,974.

The number of oligonucleotides generated for each parental variant bearsa relationship to the total number of resulting crossovers in thechimeric molecule that is ultimately created. For example, threeparental nucleotide sequence variants might be provided to undergo aligation reaction in order to find a chimeric variant having, forexample, greater activity at high temperature. As one example, a set of50 oligonucleotide sequences can be generated corresponding to eachportions of each parental variant. Accordingly, during the ligationreassembly process there could be up to 50 crossover events within eachof the chimeric sequences. The probability that each of the generatedchimeric polynucleotides will contain oligonucleotides from eachparental variant in alternating order is very low. If eacholigonucleotide fragment is present in the ligation reaction in the samemolar quantity it is likely that in some positions oligonucleotides fromthe same parental polynucleotide will ligate next to one another andthus not result in a crossover event. If the concentration of eacholigonucleotide from each parent is kept constant during any ligationstep in this example, there is a ⅓ chance (assuming 3 parents) that anoligonucleotide from the same parental variant will ligate within thechimeric sequence and produce no crossover.

Accordingly, a probability density function (PDF) can be determined topredict the population of crossover events that are likely to occurduring each step in a ligation reaction given a set number of parentalvariants, a number of oligonucleotides corresponding to each variant,and the concentrations of each variant during each step in the ligationreaction. The statistics and mathematics behind determining the PDF isdescribed below. By utilizing these methods, one can calculate such aprobability density function, and thus enrich the chimeric progenypopulation for a predetermined number of crossover events resulting froma particular ligation reaction. Moreover, a target number of crossoverevents can be predetermined, and the system then programmed to calculatethe starting quantities of each parental oligonucleotide during eachstep in the ligation reaction to result in a probability densityfunction that centers on the predetermined number of crossover events.These methods are directed to the use of repeated cycles of reductivereassortment, recombination and selection that allow for the directedmolecular evolution of a nucleic acid encoding a polypeptide throughrecombination. This system allows generation of a large population ofevolved chimeric sequences, wherein the generated population issignificantly enriched for sequences that have a predetermined number ofcrossover events. A crossover event is a point in a chimeric sequencewhere a shift in sequence occurs from one parental variant to anotherparental variant. Such a point is normally at the juncture of whereoligonucleotides from two parents are ligated together to form a singlesequence. The method allows calculation of the correct concentrations ofoligonucleotide sequences so that the final chimeric population ofsequences is enriched for the chosen number of crossover events. Thisprovides more control over choosing chimeric variants having apredetermined number of crossover events.

In addition, these methods provide a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. By using the methods described herein, the population ofchimerics molecules can be enriched for those variants that have aparticular number of crossover events. Thus, although one can stillgenerate 10¹³ chimeric molecules during a reaction, each of themolecules chosen for further analysis most likely has, for example, onlythree crossover events. Because the resulting progeny population can beskewed to have a predetermined number of crossover events, theboundaries on the functional variety between the chimeric molecules isreduced. This provides a more manageable number of variables whencalculating which oligonucleotide from the original parentalpolynucleotides might be responsible for affecting a particular trait.

In some embodiments, the method creates a chimeric progenypolynucleotide sequence by creating oligonucleotides corresponding tofragments or portions of each parental sequence. Each oligonucleotide insome embodiments includes a unique region of overlap so that mixing theoligonucleotides together results in a new variant that has eacholigonucleotide fragment assembled in the correct order. See also U.S.Pat. Nos. 6,773,900; 6,740,506; 6,713,282; 6,635,449; 6,605,449;6,537,776; 6,361,974.

Determining Crossover Events

Embodiments of the invention include a system and software that receivea desired crossover probability density function (PDF), the number ofparent genes to be reassembled, and the number of fragments in thereassembly as inputs. The output of this program is a “fragment PDF”that can be used to determine a recipe for producing reassembled genes,and the estimated crossover PDF of those genes. The processing describedherein is in some embodiments performed in MATLAB™ (The Mathworks,Natick, Mass.) a programming language and development environment fortechnical computing.

Iterative Processes

Any process in accordance with the invention can be iterativelyrepeated, such as a nucleic acid encoding an altered or new aldolasephenotype, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme in accordance with the invention, can be identified, re-isolated,again modified, re-tested for activity. This process can be iterativelyrepeated until a desired phenotype is engineered. For example, an entirebiochemical anabolic or catabolic pathway can be engineered into a cell,including, such as aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzyme, activity.

Similarly, if it is determined that a particular oligonucleotide has noaffect at all on the desired trait (such as a new aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzyme phenotype), itcan be removed as a variable by synthesizing larger parentaloligonucleotides that include the sequence to be removed. Becauseincorporating the sequence within a larger sequence prevents anycrossover events, there will no longer be any variation of this sequencein the progeny polynucleotides. This iterative practice of determiningwhich oligonucleotides are most related to the desired trait, and whichare unrelated, allows more efficient exploration all of the possibleprotein variants that might be provide a particular trait or activity.

In Vivo Shuffling

In various embodiments, in vivo shuffling of molecules is used inmethods in accordance with the invention to provide variants ofpolypeptides in accordance with the invention, such as antibodies inaccordance with the invention or aldolases in accordance with theinvention, such as pyruvate aldolase, HMG and/or KHG aldolase enzymes,and the like. In vivo shuffling can be performed utilizing the naturalproperty of cells to recombine multimers. While recombination in vivohas provided the major natural route to molecular diversity, geneticrecombination remains a relatively complex process that involves 1) therecognition of homologies; 2) strand cleavage, strand invasion, andmetabolic steps leading to the production of recombinant chiasma; andfinally 3) the resolution of chiasma into discrete recombined molecules.The formation of the chiasma requires the recognition of homologoussequences.

In other embodiments, the invention includes a method for producing ahybrid polynucleotide from at least a first polynucleotide and a secondpolynucleotide. In some embodiments, the invention can be used toproduce a hybrid polynucleotide by introducing at least a firstpolynucleotide and a second polynucleotide (such as one, or both, beingan aldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme-encoding sequence in accordance with the invention) which shareat least one region of partial sequence homology into a suitable hostcell. The regions of partial sequence homology promote processes whichresult in sequence reorganization producing a hybrid polynucleotide. Theterm “hybrid polynucleotide”, as used herein, is any nucleotide sequencewhich results from the method of the present invention and containssequence from at least two original polynucleotide sequences. Suchhybrid polynucleotides can result from intermolecular recombinationevents which promote sequence integration between DNA molecules. Inaddition, such hybrid polynucleotides can result from intramolecularreductive reassortment processes which utilize repeated sequences toalter a nucleotide sequence within a DNA molecule.

In some embodiments, vivo reassortment focuses on “inter-molecular”processes collectively referred to as “recombination”; which inbacteria, is generally viewed as a “RecA-dependent” phenomenon. In someembodiments, the invention can rely on recombination processes of a hostcell to recombine and re-assort sequences, or the cells' ability tomediate reductive processes to decrease the complexity of quasi-repeatedsequences in the cell by deletion. This process of “reductivereassortment” occurs by an “intra-molecular”, RecA-independent process.

In other embodiments of the invention, novel polynucleotides can begenerated by the process of reductive reassortment. The method involvesthe generation of constructs containing consecutive sequences (originalencoding sequences), their insertion into an appropriate vector andtheir subsequent introduction into an appropriate host cell. Thereassortment of the individual molecular identities occurs bycombinatorial processes between the consecutive sequences in theconstruct possessing regions of homology, or between quasi-repeatedunits. The reassortment process recombines and/or reduces the complexityand extent of the repeated sequences and results in the production ofnovel molecular species. Various treatments may be applied to enhancethe rate of reassortment. These could include treatment withultra-violet light, or DNA damaging chemicals and/or the use of hostcell lines displaying enhanced levels of “genetic instability”. Thus thereassortment process may involve homologous recombination or the naturalproperty of quasi-repeated sequences to direct their own evolution.

Repeated or “quasi-repeated” sequences play a role in geneticinstability. In some embodiments, “quasi-repeats” are repeats that arenot restricted to their original unit structure. Quasi-repeated unitscan be presented as an array of sequences in a construct; consecutiveunits of similar sequences. Once ligated, the junctions between theconsecutive sequences become essentially invisible and thequasi-repetitive nature of the resulting construct is now continuous atthe molecular level. The deletion process the cell performs to reducethe complexity of the resulting construct operates between thequasi-repeated sequences. The quasi-repeated units provide a practicallylimitless repertoire of templates upon which slippage events can occur.In some embodiments, the constructs containing the quasi-repeats thuseffectively provide sufficient molecular elasticity that deletion (andpotentially insertion) events can occur virtually anywhere within thequasi-repetitive units.

When the quasi-repeated sequences are all ligated in the sameorientation, for instance head to tail or vice versa, the cell cannotdistinguish individual units. Consequently, the reductive process canoccur throughout the sequences. In contrast, when for example, the unitsare presented head to head, rather than head to tail, the inversiondelineates the endpoints of the adjacent unit so that deletion formationwill favor the loss of discrete units. Thus, it is preferable with thepresent method that the sequences are in the same orientation. Randomorientation of quasi-repeated sequences will result in the loss ofreassortment efficiency, while consistent orientation of the sequenceswill offer the highest efficiency. However, while having fewer of thecontiguous sequences in the same orientation decreases the efficiency,it may still provide sufficient elasticity for the effective recovery ofnovel molecules. Constructs can be made with the quasi-repeatedsequences in the same orientation to allow higher efficiency.

Sequences can be assembled in a head to tail orientation using any of avariety of methods, including the following:

-   a) Primers that include a poly-A head and poly-T tail which when    made single-stranded would provide orientation can be utilized. This    is accomplished by having the first few bases of the primers made    from RNA and hence easily removed RNaseH.-   b) Primers that include unique restriction cleavage sites can be    utilized. Multiple sites, a battery of unique sequences and repeated    synthesis and ligation steps would be required.-   c) The inner few bases of the primer could be thiolated and an    exonuclease used to produce properly tailed molecules.

In some embodiments, the recovery of the re-assorted sequences relies onthe identification of cloning vectors with a reduced repetitive index(RI). The re-assorted encoding sequences can then be recovered byamplification. The products are re-cloned and expressed. The recovery ofcloning vectors with reduced RI can be affected by:

1) The use of vectors only stably maintained when the construct isreduced in complexity.

2) The physical recovery of shortened vectors by physical procedures. Inthis case, the cloning vector would be recovered using standard plasmidisolation procedures and size fractionated on either an agarose gel, orcolumn with a low molecular weight cut off utilizing standardprocedures.

3) The recovery of vectors containing interrupted genes which can beselected when insert size decreases.

4) The use of direct selection techniques with an expression vector andthe appropriate selection.

Encoding sequences (for example, genes) from related organisms maydemonstrate a high degree of homology and encode quite diverse proteinproducts. These types of sequences are particularly useful in thepresent invention as quasi-repeats. However, while the examplesillustrated below demonstrate the reassortment of nearly identicaloriginal encoding sequences (quasi-repeats), this process is not limitedto such nearly identical repeats.

The following example demonstrates an exemplary method in accordancewith the invention. Encoding nucleic acid sequences (quasi-repeats)derived from three (3) unique species are described. Each sequenceencodes a protein with a distinct set of properties. Each of thesequences differs by a single or a few base pairs at a unique positionin the sequence. The quasi-repeated sequences are separately orcollectively amplified and ligated into random assemblies such that allpossible permutations and combinations are available in the populationof ligated molecules. The number of quasi-repeat units can be controlledby the assembly conditions. The average number of quasi-repeated unitsin a construct is defined as the repetitive index (RI).

Once formed, the constructs may, or may not be size fractionated on anagarose gel according to published protocols, inserted into a cloningvector and transfected into an appropriate host cell. The cells are thenpropagated and “reductive reassortment” is effected. The rate of thereductive reassortment process may be stimulated by the introduction ofDNA damage if desired. Whether the reduction in RI is mediated bydeletion formation between repeated sequences by an “intra-molecular”mechanism, or mediated by recombination-like events through“inter-molecular” mechanisms is immaterial. The end result is areassortment of the molecules into all possible combinations.

Optionally, the method comprises the additional step of screening thelibrary members of the shuffled pool to identify individual shuffledlibrary members having the ability to bind or otherwise interact, orcatalyze a particular reaction (such as catalytic domain of an enzyme)with a predetermined macromolecule, such as for example a proteinaceousreceptor, an oligosaccharide, virion, or other predetermined compound orstructure.

The polypeptides that are identified from such libraries can be used fortherapeutic, diagnostic, research and related purposes (such ascatalysts, solutes for increasing osmolarity of an aqueous solution andthe like) and/or can be subjected to one or more additional cycles ofshuffling and/or selection.

In other embodiments, it is envisioned that prior to or duringrecombination or reassortment, polynucleotides generated by the methodin accordance with the invention can be subjected to agents or processeswhich promote the introduction of mutations into the originalpolynucleotides. The introduction of such mutations would increase thediversity of resulting hybrid polynucleotides and polypeptides encodedtherefrom. The agents or processes which promote mutagenesis caninclude, but are not limited to: (+)-CC-1065, or a synthetic analog suchas (+)-CC-1065-(N3-Adenine (See Sun and Hurley, (1992); an N-acetylatedor deacetylated 4′-fluoro-4-aminobiphenyl adduct capable of inhibitingDNA synthesis (See, for example, van de Poll et al. (1992)); or aN-acetylated or deacetylated 4-aminobiphenyl adduct capable ofinhibiting DNA synthesis (See also, van de Poll et al. (1992), pp.751-758); trivalent chromium, a trivalent chromium salt, a polycyclicaromatic hydrocarbon (PAH) DNA adduct capable of inhibiting DNAreplication, such as 7-bromomethyl-benz[α]anthracene (“BMA”),tris(2,3-dibromopropyl)phosphate (“Tris-BP”),1,2-dibromo-3-chloropropane (“DBCP”), 2-bromoacrolein (2BA),benzo[α]pyrene-7,8-dihydrodiol-9-10-epoxide (“BPDE”), a platinum(II)halogen salt, N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline(“N-hydroxy-IQ”) andN-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-f]-pyridine(“N-hydroxy-PhIP”). Exemplary means for slowing or halting PCRamplification consist of UV light (+)-CC-1065 and(+)-CC-1065-(N3-Adenine). Particularly encompassed means are DNA adductsor polynucleotides comprising the DNA adducts from the polynucleotidesor polynucleotides pool, which can be released or removed by a processincluding heating the solution comprising the polynucleotides prior tofurther processing.

In other embodiments the invention is directed to a method of producingrecombinant proteins having biological activity by treating a samplecomprising double-stranded template polynucleotides encoding a wild-typeprotein under conditions according to the invention which provide forthe production of hybrid or re-assorted polynucleotides.

Producing Sequence Variants

The invention also provides additional methods for making sequencevariants of the nucleic acid (such as aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzyme) sequences inaccordance with the invention. In some embodiments, the invention alsoprovides additional methods for isolating aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzymes using the nucleicacids and polypeptides in accordance with the invention. In someembodiments, the invention provides for variants of an aldolase, such aspyruvate aldolase, HMG and/or KHG aldolase enzyme coding sequence (suchas a gene, cDNA or message) in accordance with the invention, which canbe altered by any means, including, such as random or stochasticmethods, or, non-stochastic, or “directed evolution,” methods, asdescribed above.

The isolated variants may be naturally occurring. Variant can also becreated in vitro. Variants may be created using genetic engineeringtechniques such as site directed mutagenesis, random chemicalmutagenesis, Exonuclease III deletion procedures, and standard cloningtechniques. Alternatively, such variants, fragments, analogs, orderivatives may be created using chemical synthesis or modificationprocedures. Other methods of making variants are also familiar to thoseskilled in the art. These include procedures in which nucleic acidsequences obtained from natural isolates are modified to generatenucleic acids which encode polypeptides having characteristics whichenhance their value in industrial or laboratory applications. In suchprocedures, a large number of variant sequences having one or morenucleotide differences with respect to the sequence obtained from thenatural isolate are generated and characterized. These nucleotidedifferences can result in amino acid changes with respect to thepolypeptides encoded by the nucleic acids from the natural isolates.

For example, variants may be created using error prone PCR. In someembodiments of error prone PCR, the PCR is performed under conditionswhere the copying fidelity of the DNA polymerase is low, such that ahigh rate of point mutations is obtained along the entire length of thePCR product. Error prone PCR is described, such as in Leung, D. W. etal., (1989) Technique 1:11-15; and Caldwell, R. C. & Joyce, G. F.,(1992) PCR Methods Applic. 2:28-33. Briefly, in such procedures, nucleicacids to be mutagenized are mixed with PCR primers, reaction buffer,MgCl₂, MnCl₂, Taq polymerase and an appropriate concentration of dNTPsfor achieving a high rate of point mutation along the entire length ofthe PCR product. For example, the reaction may be performed using 20fmoles of nucleic acid to be mutagenized, 30 pmole of each PCR primer, areaction buffer comprising 50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01%gelatin, 7 mM MgCl₂, 0.5 mM MnCl₂, 5 units of Taq polymerase, 0.2 mMdGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP. PCR may be performed for 30cycles of 94° C. for 1 minute, 45° C. for 1 minute, and 72° C. for 1minute. However, it will be appreciated that these parameters may bevaried as appropriate. The mutagenized nucleic acids are cloned into anappropriate vector and the activities of the polypeptides encoded by themutagenized nucleic acids are evaluated.

In some embodiments, variants are created using oligonucleotide directedmutagenesis to generate site-specific mutations in any cloned DNA ofinterest. Oligonucleotide mutagenesis is described, such as inReidhaar-Olson (1988) Science 241:53-57. Briefly, in such procedures aplurality of double stranded oligonucleotides bearing one or moremutations to be introduced into the cloned DNA are synthesized andinserted into the cloned DNA to be mutagenized. In some embodiments,clones containing the mutagenized DNA are recovered, expressed, and theactivities of the polypeptide encoded therein assessed.

Another method for generating variants is assembly PCR. Assembly PCRinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction. Assembly PCR is described in the art, such as inU.S. Pat. No. 5,965,408.

In some embodiments, sexual PCR mutagenesis is an exemplary method ofgenerating variants in accordance with the invention. In someembodiments of sexual PCR mutagenesis forced homologous recombinationoccurs between DNA molecules of different but highly related DNAsequence in vitro, as a result of random fragmentation of the DNAmolecule based on sequence homology, followed by fixation of thecrossover by primer extension in a PCR reaction. Sexual PCR mutagenesisis described, such as in Stemmer (1994) Proc. Natl. Acad. Sci. USA91:10747-10751. Briefly, in such procedures a plurality of nucleic acidsto be recombined are digested with DNase to generate fragments having anaverage size of 50-200 nucleotides. Fragments of the desired averagesize are purified and resuspended in a PCR mixture. PCR is conductedunder conditions which facilitate recombination between the nucleic acidfragments. For example, PCR may be performed by resuspending thepurified fragments at a concentration of 10-30 ng/μl in a solution of0.2 mM of each dNTP, 2.2 mM MgCl₂, 50 mM KCL, 10 mM Tris HCl, pH 9.0,and 0.1% Triton X-100. 2.5 units of Taq polymerase per 100 μl ofreaction mixture is added and PCR is performed using the followingregime: 94° C. for 60 seconds, 94° C. for 30 seconds, 50-55° C. for 30seconds, 72° C. for 30 seconds (30-45 times) and 72° C. for 5 minutes.However, it will be appreciated that these parameters may be varied asappropriate. In some embodiments, oligonucleotides may be included inthe PCR reactions. In other embodiments, the Klenow fragment of DNApolymerase I may be used in a first set of PCR reactions and Taqpolymerase may be used in a subsequent set of PCR reactions. Recombinantsequences are isolated and the activities of the polypeptides theyencode are assessed.

In some embodiments, variants are created by in vivo mutagenesis. Insome embodiments, random mutations in a sequence of interest aregenerated by propagating the sequence of interest in a bacterial strain,such as an E. coli strain, which carries mutations in one or more of theDNA repair pathways. Such “mutator” strains have a higher randommutation rate than that of a wild-type parent. Propagating the DNA inone of these strains will eventually generate random mutations withinthe DNA. Mutator strains suitable for use for in vivo mutagenesis aredescribed in PCT Publication No. WO 91/16427, published Oct. 31, 1991,entitled “Methods for Phenotype Creation from Multiple GenePopulations”.

Variants may also be generated using cassette mutagenesis. In cassettemutagenesis a small region of a double stranded DNA molecule is replacedwith a synthetic oligonucleotide “cassette” that differs from the nativesequence. The oligonucleotide often contains completely and/or partiallyrandomized native sequence.

Recursive ensemble mutagenesis may also be used to generate variants.Recursive ensemble mutagenesis is an algorithm for protein engineering(protein mutagenesis) developed to produce diverse populations ofphenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Recursive ensemblemutagenesis is described, such as in Arkin (1992) Proc. Natl. Acad. Sci.USA 89:7811-7815.

In some embodiments, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is described,such as in Delegrave (1993) Biotechnology Res. 11:1548-1552. Random andsite-directed mutagenesis are described, such as in Arnold (1993)Current Opinion in Biotechnology 4:450-455.

In some embodiments, the variants are created using shuffling procedureswherein portions of a plurality of nucleic acids which encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences which encode chimeric polypeptides as described in U.S. Pat.No. 5,965,408, filed Jul. 9, 1996, entitled, “Method of DNA Reassemblyby Interrupting Synthesis” and U.S. Pat. No. 5,939,250, filed May 22,1996, entitled, “Production of Enzymes Having Desired Activities byMutagenesis.

The variants of the polypeptides in accordance with the invention may bevariants in which one or more of the amino acid residues of thepolypeptides of the sequences in accordance with the invention aresubstituted with a conserved or non-conserved amino acid residue (insome embodiments, a conserved amino acid residue) and such substitutedamino acid residue may or may not be one encoded by the genetic code.

In some embodiments, conservative substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. In some embodiments, conservative substitutions inaccordance with the invention comprise the following replacements:replacements of an aliphatic amino acid such as Alanine, Valine, Leucineand Isoleucine with another aliphatic amino acid; replacement of aSerine with a Threonine or vice versa; replacement of an acidic residuesuch as Aspartic acid and Glutamic acid with another acidic residue;replacement of a residue bearing an amide group, such as Asparagine andGlutamine, with another residue bearing an amide group; exchange of abasic residue such as Lysine and Arginine with another basic residue;and replacement of an aromatic residue such as Phenylalanine, Tyrosinewith another aromatic residue.

Other variants are those in which one or more of the amino acid residuesof a polypeptide in accordance with the invention includes a substituentgroup. In some embodiments, other variants are those in which thepolypeptide is associated with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol). Additional variants are those in which additional amino acidsare fused to the polypeptide, such as a leader sequence, a secretorysequence, a proprotein sequence or a sequence which facilitatespurification, enrichment, or stabilization of the polypeptide.

In some embodiments, the fragments, derivatives and analogs retain thesame biological function or activity as the polypeptides in accordancewith the invention and sequences substantially identical thereto. Inother embodiments, the fragment, derivative, or analog includes aproprotein, such that the fragment, derivative, or analog can beactivated by cleavage of the proprotein portion to produce an activepolypeptide.

Optimizing Codons to Achieve High Levels of Protein Expression in HostCells

The invention provides methods for modifying aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase, enzyme-encoding nucleic acidsto modify (such as optimize) codon usage. In some embodiments, theinvention provides methods for modifying codons in a nucleic acidencoding an aldolase, such as pyruvate aldolase, HMG and/or KHG aldolaseenzyme to increase or decrease its expression in a host cell. In someembodiments, the invention also provides nucleic acids encoding analdolase, such as pyruvate aldolase, HMG and/or KHG aldolase enzymemodified to increase its expression in a host cell, aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzyme so modified,and methods of making the modified aldolase, such as pyruvate aldolase,such as HMG and/or KHG aldolase enzymes. The method comprisesidentifying a “non-preferred” or a “less preferred” codon in aldolase,such as pyruvate aldolase, such as HMG and/or KHG aldolase,enzyme-encoding nucleic acid and replacing one or more of thesenon-preferred or less preferred codons with a “preferred codon” encodingthe same amino acid as the replaced codon and at least one non-preferredor less preferred codon in the nucleic acid has been replaced by apreferred codon encoding the same amino acid. A preferred codon is acodon over-represented in coding sequences in genes in the host cell anda non-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell.

Host cells for expressing the nucleic acids, expression cassettes andvectors in accordance with the invention include bacteria, yeast, fungi,plant cells, insect cells and mammalian cells (see discussion, above).Thus, the invention provides methods for optimizing codon usage in allof these cells, codon-altered nucleic acids and polypeptides made by thecodon-altered nucleic acids. Exemplary host cells include gram negativebacteria, such as Escherichia coli; gram positive bacteria, such asStreptomyces sp., Lactobacillus gasseri, Lactococcus lactis, Lactococcuscremoris, Bacillus subtilis, Bacillus cereus. Exemplary host cells alsoinclude eukaryotic organisms, such as various yeast, such asSaccharomyces sp., including Saccharomyces cerevisiae,Schizosaccharomyces pombe, Pichia pastoris, and Kluyveromyces lactis,Hansenula polymorpha, Aspergillus niger, and mammalian cells and celllines and insect cells and cell lines. Thus, the invention also includesnucleic acids and polypeptides optimized for expression in theseorganisms and species.

For example, the codons of a nucleic acid encoding an aldolase, such aspyruvate aldolase, HMG and/or KHG aldolase enzyme isolated from abacterial cell are modified such that the nucleic acid is optimallyexpressed in a bacterial cell different from the bacteria from which thealdolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme was derived, a yeast, a fungi, a plant cell, an insect cell or amammalian cell. Methods for optimizing codons are well known in the art,see U.S. Pat. No. 5,795,737; Baca (2000) Int. J. Parasitol. 30:113-118;Hale (1998) Protein Expr. Purif. 12:185-188; Narum (2001) Infect. Immun.69:7250-7253. See also Narum (2001) Infect. Immun 69:7250-7253,describing optimizing codons in mouse systems; Outchkourov (2002)Protein Expr. Purif. 24:18-24, describing optimizing codons in yeast;Feng (2000) Biochemistry 39:15399-15409, describing optimizing codons inE. coli; Humphreys (2000) Protein Expr. Purif. 20:252-264, describingoptimizing codon usage that affects secretion in E. coli.

Transgenic Non-Human Animals

The invention provides transgenic non-human animals comprising a nucleicacid, a polypeptide (such as an aldolase, such as pyruvate aldolase, HMGand/or KHG aldolase enzyme), an expression cassette or vector or atransfected or transformed cell in accordance with the invention. Insome embodiments, the invention also provides methods of making andusing these transgenic non-human animals.

The transgenic non-human animals can be, such as dogs, goats, rabbits,sheep, horses, fish, pigs (including all swine, hogs and relatedanimals), cows, rats and mice, comprising the nucleic acids inaccordance with the invention. These animals can be used, such as invivo models to study aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzyme, activity, or, as models to screen for agentsthat change the aldolase, such as pyruvate aldolase, such as HMG and/orKHG aldolase enzyme, activity in vivo. The coding sequences for thepolypeptides to be expressed in the transgenic non-human animals can bedesigned to be constitutive, or, under the control of tissue-specific,developmental-specific or inducible transcriptional regulatory factors.

Transgenic non-human animals can be designed and generated using anymethod known in the art; see U.S. Pat. Nos. 6,211,428; 6,187,992;6,156,952; 6,118,044; 6,111,166; 6,107,541; 5,959,171; 5,922,854;5,892,070; 5,880,327; 5,891,698; 5,639,940; 5,573,933; 5,387,742;5,087,571, describing making and using transformed cells and eggs andtransgenic mice, rats, rabbits, sheep, pigs, chickens, goats, fish andcows. See also, such as Pollock (1999) J. Immunol. Methods 231:147-157,describing the production of recombinant proteins in the milk oftransgenic dairy animals; Baguisi (1999) Nat. Biotechnol. 17:456-461,demonstrating the production of transgenic goats. U.S. Pat. No.6,211,428, describes making and using transgenic non-human mammals whichexpress in their brains a nucleic acid construct comprising a DNAsequence. U.S. Pat. No. 5,387,742, describes injecting clonedrecombinant or synthetic DNA sequences into fertilized mouse eggs,implanting the injected eggs in pseudo-pregnant females, and growing toterm transgenic mice. U.S. Pat. No. 6,187,992, describes making andusing a transgenic mouse.

“Knockout animals” can also be used to practice the methods inaccordance with the invention. For example, in some embodiments, thetransgenic or modified animals in accordance with the invention comprisea “knockout animal,” such as a “knockout mouse,” engineered not toexpress an endogenous gene, which is replaced with a gene expressing analdolase, such as pyruvate aldolase, HMG and/or KHG aldolase enzyme inaccordance with the invention, or, a fusion protein comprising analdolase, such as pyruvate aldolase, HMG and/or KHG aldolase enzyme inaccordance with the invention.

Transgenic Plants and Seeds

The invention provides transgenic plants and seeds comprising a nucleicacid, a polypeptide (such as an aldolase, such as pyruvate aldolase, HMGand/or KHG aldolase enzyme), an expression cassette or vector or atransfected or transformed cell in accordance with the invention. Theinvention also provides plant products or byproducts, such as fruits,oils, seeds, leaves, extracts and the like, including any plant part,comprising a nucleic acid and/or a polypeptide (such as a xylanase) ofthe invention, such as wherein the nucleic acid or polypeptide of theinvention is heterologous to the plant, plant part, seed etc. Thetransgenic plant (which includes plant parts, fruits, seeds etc.) can bedicotyledonous (a dicot) or monocotyledonous (a monocot). In someembodiments, the invention also provides methods of making and usingthese transgenic plants and seeds. The transgenic plant or plant cellexpressing a polypeptide of the present invention may be constructed inaccordance with any method known in the art. See, for example, U.S. Pat.No. 6,309,872.

Nucleic acids and expression constructs in accordance with the inventioncan be introduced into a plant cell by any means. For example, nucleicacids or expression constructs can be introduced into the genome of adesired plant host, or, the nucleic acids or expression constructs canbe episomes. Introduction into the genome of a desired plant can be suchthat the host's aldolase, such as pyruvate aldolase, such as HMG and/orKHG aldolase enzyme production is regulated by endogenoustranscriptional or translational control elements. In some embodiments,the invention also provides “knockout plants” where insertion of genesequence by, such as homologous recombination, has disrupted theexpression of the endogenous gene. Means to generate “knockout” plantsare well-known in the art, see Strepp (1998) Proc Natl. Acad. Sci. USA95:4368-4373; Miao (1995) Plant J 7:359-365. See discussion ontransgenic plants, below.

The nucleic acids in accordance with the invention can be used to conferdesired traits on essentially any plant, such as on starch-producingplants, such as potato, tomato, soybean, beets, corn, wheat, rice,barley, and the like. Nucleic acids in accordance with the invention canbe used to manipulate metabolic pathways of a plant in order to optimizeor alter host's expression of aldolase, such as pyruvate aldolase, suchas HMG and/or KHG aldolase enzyme. The nucleic acids in accordance withthe invention can change expression or activity levels or altercharacteristics of compounds or enzymes naturally produced in a plant.Alternatively, an aldolase, such as pyruvate aldolase, HMG and/or KHGaldolase enzyme in accordance with the invention can be used inproduction of a transgenic plant to produce a compound not naturallyproduced by that plant. This can lower production costs or create anovel product.

In some embodiments, the first step in production of a transgenic plantinvolves making an expression construct for expression in a plant cell.These techniques are well known in the art. They can include selectingand cloning a promoter, a coding sequence for facilitating efficientbinding of ribosomes to mRNA and selecting the appropriate geneterminator sequences. One exemplary constitutive promoter is CaMV35S,from the cauliflower mosaic virus, which generally results in a highdegree of expression in plants. Other promoters are more specific andrespond to cues in the plant's internal or external environment. Anexemplary light-inducible promoter is the promoter from the cab gene,encoding the major chlorophyll a/b binding protein.

In some embodiments, the nucleic acid is modified to achieve greaterexpression in a plant cell. For example, a sequence in accordance withthe invention is likely to have a higher percentage of A-T nucleotidepairs compared to that seen in a plant, some of which prefer G-Cnucleotide pairs. Therefore, A-T nucleotides in the coding sequence canbe substituted with G-C nucleotides without significantly changing theamino acid sequence to enhance production of the gene product in plantcells.

Selectable marker gene can be added to the gene construct in order toidentify plant cells or tissues that have successfully integrated thetransgene. This may be necessary because achieving incorporation andexpression of genes in plant cells is a rare event, occurring in just afew percent of the targeted tissues or cells. Selectable marker genesencode proteins that provide resistance to agents that are normallytoxic to plants, such as antibiotics or herbicides. Only plant cellsthat have integrated the selectable marker gene will survive when grownon a medium containing the appropriate antibiotic or herbicide. As forother inserted genes, marker genes also require promoter and terminationsequences for proper function.

In some embodiments, making transgenic plants or seeds comprisesincorporating sequences in accordance with the invention and,optionally, marker genes into a target expression construct (such as aplasmid), along with positioning of the promoter and the terminatorsequences. This can involve transferring the modified gene into theplant through a suitable method. For example, a construct may beintroduced directly into the genomic DNA of the plant cell usingtechniques such as electroporation and microinjection of plant cellprotoplasts, or the constructs can be introduced directly to planttissue using ballistic methods, such as DNA particle bombardment. Forexample, see Christou (1997) Plant Mol. Biol. 35:197-203; Pawlowski(1996) Mol. Biotechnol. 6:17-30; Klein (1987) Nature 327:70-73; Takumi(1997) Genes Genet. Syst. 72:63-69, discussing use of particlebombardment to introduce transgenes into wheat; and Adam (1997) supra,for use of particle bombardment to introduce YACs into plant cells. Forexample, Rinehart (1997) supra, used particle bombardment to generatetransgenic cotton plants. Apparatus for accelerating particles isdescribed U.S. Pat. No. 5,015,580; and, the commercially availableBio-Rad (Biolistics) PDS-2000 particle acceleration instrument (Bio-Rad,Hercules, Calif.); see also, John, U.S. Pat. No. 5,608,148; and Ellis,U.S. Pat. No. 5,681,730, describing particle-mediated transformation ofgymnosperms.

In some embodiments, protoplasts can be immobilized and injected with anucleic acids, such as an expression construct. Although plantregeneration from protoplasts is not easy with cereals, plantregeneration is possible in legumes using somatic embryogenesis fromprotoplast derived callus. Organized tissues can be transformed withnaked DNA using gene gun technique, where DNA is coated on tungstenmicroprojectiles, shot 1/100th the size of cells, which carry the DNAdeep into cells and organelles. Transformed tissue is then induced toregenerate, usually by somatic embryogenesis. This technique has beensuccessful in several cereal species including maize and rice.

Nucleic acids, such as expression constructs, can also be introducedinto plant cells using recombinant viruses. Plant cells can betransformed using viral vectors, such as tobacco mosaic virus derivedvectors (Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta (1996)“Use of viral replicons for the expression of genes in plants,” Mol.Biotechnol. 5:209-221.

Alternatively, nucleic acids, such as an expression construct, can becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria. Agrobacteriumtumefaciens-mediated transformation techniques, including disarming anduse of binary vectors, are well described in the scientific literature.See Horsch (1984) Science 233:496-498; Fraley (1983) Proc. Natl. Acad.Sci. USA 80:4803 (1983); Gene Transfer to Plants, Potrykus, ed.(Springer-Verlag, Berlin 1995). The DNA in an A. tumefaciens cell iscontained in the bacterial chromosome as well as in another structureknown as a Ti (tumor-inducing) plasmid. The Ti plasmid contains astretch of DNA termed T-DNA (˜20 kb long) that is transferred to theplant cell in the infection process and a series of vir (virulence)genes that direct the infection process. A. tumefaciens can only infecta plant through wounds: when a plant root or stem is wounded it givesoff certain chemical signals, in response to which, the vir genes of A.tumefaciens become activated and direct a series of events necessary forthe transfer of the T-DNA from the Ti plasmid to the plant's chromosome.The T-DNA then enters the plant cell through the wound. One speculationis that the T-DNA waits until the plant DNA is being replicated ortranscribed, then inserts itself into the exposed plant DNA. In order touse A. tumefaciens as a transgene vector, the tumor-inducing section ofT-DNA have to be removed, while retaining the T-DNA border regions andthe vir genes. The transgene is then inserted between the T-DNA borderregions, where it is transferred to the plant cell and becomesintegrated into the plant's chromosomes.

The invention provides for the transformation of monocotyledonous plantsusing the nucleic acids in accordance with the invention, includingimportant cereals, see Hiei (1997) Plant Mol. Biol. 35:205-218. Seealso, Horsch, Science (1984) 233:496; Fraley (1983) Proc. Natl. Acad.Sci. USA 80:4803; Thykjaer (1997) supra; Park (1996) Plant Mol. Biol.32:1135-1148, discussing T-DNA integration into genomic DNA. See alsoD'Halluin, U.S. Pat. No. 5,712,135, describing a process for the stableintegration of a DNA comprising a gene that is functional in a cell of acereal, or other monocotyledonous plant.

In some embodiments, the third step involves selection and regenerationof whole plants capable of transmitting the incorporated target gene tothe next generation. Such regeneration techniques may use manipulationof certain phytohormones in a tissue culture growth medium. In someembodiments, the method uses a biocide and/or herbicide marker that hasbeen introduced together with the desired nucleotide sequences. Plantregeneration from cultured protoplasts is described in Evans et al.,Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp.124-176, MacMillilan Publishing Company, New York, 1983; and Binding,Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, BocaRaton, 1985. Regeneration can also be obtained from plant callus,explants, organs, or parts thereof. Such regeneration techniques aredescribed generally in Klee (1987) Ann. Rev. of Plant Phys. 38:467-486.To obtain whole plants from transgenic tissues such as immature embryos,they can be grown under controlled environmental conditions in a seriesof media containing nutrients and hormones, a process known as tissueculture. Once whole plants are generated and produce seed, evaluation ofthe progeny begins.

In some embodiments, after the expression cassette is stablyincorporated in transgenic plants, it can be introduced into otherplants by sexual crossing. Any of a number of standard breedingtechniques can be used, depending upon the species to be crossed.Because transgenic expression of the nucleic acids in accordance withthe invention leads to phenotypic changes, plants comprising therecombinant nucleic acids in accordance with the invention can besexually crossed with a second plant to obtain a final product. Thus,the seed in accordance with the invention can be derived from a crossbetween two transgenic plants in accordance with the invention, or across between a plant in accordance with the invention and anotherplant. The desired effects (such as expression of the polypeptides inaccordance with the invention to produce a plant in which floweringbehavior is altered) can be enhanced when both parental plants expressthe polypeptides (such as an aldolase, such as pyruvate aldolase, HMGand/or KHG aldolase enzyme) in accordance with the invention. Thedesired effects can be passed to future plant generations by standardpropagation means.

In some embodiments, the nucleic acids and polypeptides in accordancewith the invention are expressed in or inserted in any plant or seed.Transgenic plants in accordance with the invention can be dicotyledonousor monocotyledonous. Examples of monocot transgenic plants in accordancewith the invention are grasses, such as meadow grass (blue grass, Poa),forage grass such as festuca, lolium, temperate grass, such as Agrostis,and cereals, such as wheat, oats, rye, barley, rice, sorghum, and maize(corn). Examples of dicot transgenic plants in accordance with theinvention are tobacco, legumes, such as lupins, potato, sugar beet, pea,bean and soybean, and cruciferous plants (family Brassicaceae), such ascauliflower, rape seed, and the closely related model organismArabidopsis thaliana. Thus, the transgenic plants and seeds inaccordance with the invention include a broad range of plants,including, but not limited to, species from the genera Anacardium,Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus,Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis,Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum,Hyoscyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus,Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum,Pannisetum, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus,Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum,Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.

In alternative embodiments, the nucleic acids in accordance with theinvention are expressed in plants which contain fiber cells, including,such as cotton, silk cotton tree (Kapok, Ceiba pentandra), desertwillow, creosote bush, winterfat, balsa, ramie, kenaf, hemp, roselle,jute, sisal abaca and flax. In alternative embodiments, the transgenicplants in accordance with the invention can be members of the genusGossypium, including members of any Gossypium species, such as G.arboreum; G. herbaceum, G. barbadense, and G. hirsutum.

The invention also provides for transgenic plants to be used forproducing large amounts of the polypeptides (such as an aldolase, suchas pyruvate aldolase, HMG and/or KHG aldolase enzyme or antibody) inaccordance with the invention. For example, see Palmgren (1997) TrendsGenet. 13:348; Chong (1997) Transgenic Res. 6:289-296 (producing humanmilk protein beta-casein in transgenic potato plants using anauxin-inducible, bidirectional mannopine synthase (mas1′,2′) promoterwith Agrobacterium tumefaciens-mediated leaf disc transformationmethods).

Using known procedures, one of skill can screen for plants in accordancewith the invention by detecting the increase or decrease of transgenemRNA or protein in transgenic plants. Means for detecting andquantitation of mRNAs or proteins are well known in the art.

Polypeptides and Peptides

In some embodiments, the invention provides isolated, synthetic orrecombinant polypeptides having a sequence identity (such as at leastabout 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)sequence identity, or homology) to a sequence in accordance with theinvention, such as proteins having a sequence as set forth in SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52,SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62,SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72,SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82,SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92,SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102,SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ IDNO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130,SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ IDNO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158,SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ IDNO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186,SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ IDNO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214,SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ IDNO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242,SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ IDNO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258, SEQ ID NO:260, SEQID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQ ID NO:268, SEQ ID NO:270,SEQ ID NO:272, SEQ ID NO:274, SEQ ID NO:276, SEQ ID NO:278, SEQ IDNO:280, SEQ ID NO:282, SEQ ID NO:284, SEQ ID NO:286, SEQ ID NO:288, SEQID NO:290, SEQ ID NO:292, SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298,SEQ ID NO:300, SEQ ID NO:302, SEQ ID NO:304, SEQ ID NO:306, SEQ IDNO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326,SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, or SEQ ID NO:334 andenzymatically active fragments thereof. The percent sequence identitycan be over the full length of the polypeptide, or, the identity can beover a region of at least about 50, 60, 70, 80, 90, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700 or more residues.

Polypeptides in accordance with some embodiments of the invention canalso be shorter than the full length of the polypeptides. In otherembodiments, the invention provides polypeptides (peptides, fragments)ranging in size between about 5 and the full length of a polypeptide,such as an enzyme, such as an aldolase, such as pyruvate aldolase, HMGand/or KHG aldolase enzyme; exemplary sizes being of about 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125,150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or moreresidues, such as contiguous residues of an aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzyme in accordance with theinvention. Peptides in accordance with the invention (such as asubsequence of a polypeptide in accordance with the invention) can beuseful as, such as labeling probes, antigens (immunogens), toleragens,motifs, aldolase, such as pyruvate aldolase, such as HMG and/or KHGaldolase enzyme active sites (such as “catalytic domains”), signalsequences and/or prepro domains.

In other embodiments, polypeptides in accordance with the inventionhaving aldolase activity, such as pyruvate aldolase, such as HMG and/orKHG aldolase activity are members of a genus of polypeptides sharingspecific structural elements, such as amino acid residues, thatcorrelate with aldolase activity, including pyruvate activity such as,without limitation, HMG and/or KHG aldolase activity. These sharedstructural elements can be used for the routine generation of aldolase,such as pyruvate aldolase, such as HMG and/or KHG aldolase variants.These shared structural elements of aldolase, such as pyruvate aldolase,such as HMG and/or KHG aldolase enzymes in accordance with the inventioncan be used as guidance for the routine generation of aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzymes variantswithin the scope of the genus of polypeptides in accordance with theinvention.

As used herein, the terms “aldolase, such as pyruvate aldolase, such asHMG and/or KHG aldolase” encompass any polypeptide or enzymes capable ofcatalyzing the aldol addition reaction or the retro-aldol reaction (suchas polypeptides in accordance with the invention, see also Table 1 andExamples 4, 5 and 6, below), or any modification of a carbon-carbon bondcontaining material, such as in the production of R-2-hydroxy2-(indol-3ylmethyl)-4-keto glutaric acid (R-MP) and certainstereoisomers of monatin, such as R,R and S,R monatin, and saltsthereof.

Polypeptides in accordance with some embodiments of the inventioncatalyze the formation of carbon-carbon bonds in an aldol reaction andhave the ability to utilize pyruvate or phosphoenolpyruvate as thenucleophilic component in the synthesis of a 4-hydroxy-2-ketobutyrateframework as shown in the general scheme below.

Without being bound by theory, it is believed that the conservedfour-carbon fragment prepared in all pyruvate aldolase-catalyzedcondensations is both densely and differentially functionalized.Moreover, in each adduct, four different oxidation states of carbon arecontained in four contiguous carbons. The framework prepared by pyruvatealdolases thus allows the preparation of α-amino-γ-hydroxycarboxylicacids, β-hydroxycarboxylic acids, α,γ-dihydroxycarboxylic acids, and2-deoxyaldose sugars as shown in the scheme below.

Therefore, pyruvate aldolases in accordance with some embodiments of theinvention can be synthetically versatile and can be used in thepreparation of a wide range of products for use in animal feeds, humanfoods, industrial processes, and pharmaceuticals (see, for example,Gijsen, H. J. M. et al., Recent Advances in the Chemoenzymatic Synthesisof Carbohydrates and Carbohydrate Mimetics, Chem. Rev. 1996, 96,443-473; Henderson, D. P. et al. J. Org. Chem., StereospecificPreparation of the N-Terminal Amino Acid Moiety of Nikkomycins KC and KZvia a Multiple Enzyme Synthesis, 1997, 62, 7910-7911; Wymer, N. & Toone,E. J. Enzyme-catalyzed Synthesis of Carbohydrates. Current Opin.Chemical Biology, 2000, 4, 110-119).

Polypeptides in accordance with some embodiments of the invention mayhave more than one type of enzymatic activity, specifically aldolaseactivity and an additional activity, for example, as set forth in Table1, below. For example, a polypeptide in accordance with the inventioncan have aldolase activity, pyruvate aldolase, HMG and/or KHG aldolaseactivity. Additionally, the polypeptide may have, or may be thought tohave, additional enzyme activity based on its EC classification. Table 1includes the column “Predicted EC Number”. An EC number is the numberassigned to a type of enzyme according to a scheme of standardizedenzyme nomenclature developed by the Enzyme Commission of theNomenclature Committee of the International Union of Biochemistry andMolecular Biology (IUBMB). The results in the “Predicted EC Number”column are determined by a BLAST search against the Kegg (KyotoEncyclopedia of Genes and Genomes) database. If the top BLAST match(also called a “hit”) has an Evalue equal to or less than e⁻⁶, the ECnumber assigned to the top match is entered into the table. The ECnumber of the top hit is used as a guide to what the EC number of thesequence of the invention might be. In instances where only a partial ECnumber is given, only a broad classification could be assigned based onthe top hit. For instance, in the first row, for SEQ ID NO:2, encoded bySEQ ID NO:1, the Predicted EC Number is listed as “2 . . . ”. Therefore,the classification assigned is broadly a transferase. For SEQ ID NO:26,encoded by SEQ ID NO:25, the most specific classification that could beassigned based on the top hit is as an aldehyde-lyase.

TABLE 1 SignalP Signal Predicted (AA = SEQ ID Aldolase EC Amino NO:Activity subclass Number Acid) Source 1, 2 Aldolase HMG 2 . . . Bacteria3, 4 Aldolase HMG 2 . . . Unknown 5, 6 Aldolase HMG 2 . . . Unknown 7, 8Aldolase HMG 2 . . . Unknown  9, 10 Aldolase HMG 2 . . . Unknown 11, 12Aldolase HMG 2 . . . Unknown 13, 14 Aldolase HMG 2 . . . Unknown 15, 16Aldolase HMG 2 . . . Unknown 17, 18 Aldolase HMG 2 . . . Unknown 19, 20Aldolase HMG 2 . . . Unknown 21, 22 Aldolase HMG Unknown 23, 24 AldolaseHMG 2 . . . Unknown 25, 26 Aldolase HMG 4.1.2. Unknown 27, 28 AldolaseHMG 2 . . . Unknown 29, 30 Aldolase HMG 2 . . . Unknown 31, 32 AldolaseHMG 2 . . . Unknown 33, 34 Aldolase HMG 2 . . . Unknown 35, 36 AldolaseHMG 2 . . . Unknown 37, 38 Aldolase HMG 2 . . . Unknown 39, 40 AldolaseHMG 2 . . . Unknown 41, 42 Aldolase HMG 2 . . . Unknown 43, 44 AldolaseHMG 2 . . . Unknown 45, 46 Aldolase HMG 2 . . . Unknown 47, 48 AldolaseHMG 2 . . . Unknown 49, 50 Aldolase HMG 2 . . . Unknown 51, 52 AldolaseHMG 2 . . . Unknown 53, 54 Aldolase HMG 2 . . . Unknown 55, 56 AldolaseHMG 2 . . . Unknown 57, 58 Aldolase HMG 2 . . . Unknown 59, 60 AldolaseHMG 2 . . . Unknown 61, 62 Aldolase HMG 2 . . . Unknown 63, 64 AldolaseHMG 2 . . . Unknown 65, 66 Aldolase HMG 2 . . . AA1-27 Unknown 67, 68Aldolase HMG 2 . . . Unknown 69, 70 Aldolase HMG 2 . . . Unknown 71, 72Aldolase HMG 2 . . . Unknown 73, 74 Aldolase HMG 2 . . . Unknown 75, 76Aldolase HMG 2 . . . Unknown 77, 78 Aldolase HMG 2 . . . Unknown 79, 80Aldolase HMG 2 . . . Unknown 81, 82 Aldolase HMG 2 . . . Unknown 83, 84Aldolase HMG 2 . . . Unknown 85, 86 Aldolase HMG 2 . . . Unknown 87, 88Aldolase HMG 2 . . . Unknown 89, 90 Aldolase HMG 2 . . . Unknown 91, 92Aldolase HMG 2 . . . Unknown 93, 94 Aldolase HMG 2 . . . Unknown 95, 96Aldolase HMG 2 . . . Unknown 97, 98 Aldolase HMG 2 . . . Unknown  99,100 Aldolase HMG 2 . . . Unknown 101, 102 Aldolase HMG 2 . . . Unknown103, 104 Aldolase HMG 2 . . . Unknown 105, 106 Aldolase HMG 2 . . .Unknown 107, 108 Aldolase HMG 2 . . . Unknown 109, 110 Aldolase HMG 2 .. . Unknown 111, 112 Aldolase HMG 2 . . . Unknown 113, 114 Aldolase HMG2 . . . Unknown 115, 116 Aldolase HMG 2 . . . Unknown 117, 118 AldolaseHMG 2 . . . Unknown 119, 120 Aldolase HMG 2 . . . Unknown 121, 122Aldolase HMG 2 . . . Unknown 123, 124 Aldolase HMG 2 . . . Unknown 125,126 Aldolase HMG 2 . . . Unknown 127, 128 Aldolase HMG 2 . . . Unknown129, 130 Aldolase HMG 2 . . . Unknown 131, 132 Aldolase HMG 2 . . .Unknown 133, 134 Aldolase HMG 2 . . . Unknown 135, 136 Aldolase HMG 2 .. . Unknown 137, 138 Aldolase HMG 2 . . . Unknown 139, 140 Aldolase HMG2 . . . Unknown 141, 142 Aldolase HMG 2 . . . Unknown 143, 144 AldolaseHMG 2 . . . Unknown 145, 146 Aldolase HMG 2 . . . Unknown 147, 148Aldolase HMG 2 . . . Unknown 149, 150 Aldolase HMG 2 . . . Unknown 151,152 Aldolase HMG 2 . . . Unknown 153, 154 Aldolase HMG 2 . . . Unknown155, 156 Aldolase HMG 2 . . . Unknown 157, 158 Aldolase HMG 2 . . .Unknown 159, 160 Aldolase HMG 2 . . . Unknown 161, 162 Aldolase HMG 2 .. . Unknown 163, 164 Aldolase HMG 2 . . . Unknown 165, 166 Aldolase HMG2 . . . Unknown 167, 168 Aldolase HMG 2 . . . Unknown 169, 170 AldolaseHMG 2 . . . Unknown 171, 172 Aldolase HMG 2 . . . Unknown 173, 174Aldolase HMG 2 . . . Unknown 175, 176 Aldolase HMG 2 . . . Unknown 177,178 Aldolase HMG 2 . . . Unknown 179, 180 Aldolase HMG 2 . . . Unknown181, 182 Aldolase HMG 2 . . . AA1-31 Unknown 183, 184 Aldolase HMG 2 . .. Unknown 185, 186 Aldolase HMG 2 . . . Unknown 187, 188 Aldolase HMG 2. . . Unknown 189, 190 Aldolase HMG 2 . . . Unknown 191, 192 AldolaseHMG 2 . . . Unknown 193, 194 Aldolase HMG 2 . . . Unknown 195, 196Aldolase HMG 2 . . . Unknown 197, 198 Aldolase HMG 2 . . . Unknown 199,200 Aldolase HMG 2 . . . Unknown 201, 202 Aldolase HMG 2 . . . Unknown203, 204 Aldolase HMG 2 . . . Unknown 205, 206 Aldolase HMG 2 . . .Unknown 207, 208 Aldolase HMG 2 . . . Unknown 209, 210 Aldolase HMG 2 .. . Unknown 211, 212 Aldolase HMG 2 . . . Unknown 213, 214 Aldolase HMG2 . . . Unknown 215, 216 Aldolase HMG 2 . . . Unknown 217, 218 AldolaseHMG 2 . . . Unknown 219, 220 Aldolase HMG 2 . . . Unknown 221, 222Aldolase HMG 2 . . . Unknown 223, 224 Aldolase HMG 2 . . . Unknown 225,226 Aldolase HMG 2 . . . Unknown 227, 228 Aldolase HMG 2 . . . Unknown229, 230 Aldolase HMG 2 . . . Unknown 231, 232 Aldolase HMG 2 . . .Unknown 233, 234 Aldolase HMG 2 . . . Unknown 235, 236 Aldolase HMG 2 .. . Unknown 237, 238 Aldolase HMG 2 . . . Unknown 239, 240 Aldolase HMG2 . . . Unknown 241, 242 Aldolase HMG 2 . . . Unknown 243, 244 AldolaseHMG 2 . . . Unknown 245, 246 Aldolase HMG 2 . . . Unknown 247, 248Aldolase HMG 2 . . . Unknown 249, 250 Aldolase HMG 2 . . . Unknown 251,252 Aldolase HMG 2 . . . Unknown 253, 254 Aldolase HMG 2 . . . Unknown255, 256 Aldolase HMG 2 . . . Unknown 257, 258 Aldolase HMG 2 . . .Unknown 259, 260 Aldolase HMG 2 . . . AA1-18 Unknown 261, 262 AldolaseHMG 2 . . . Unknown 263, 264 Aldolase HMG 2 . . . Unknown 265, 266Aldolase HMG 2 . . . Unknown 267, 268 Aldolase HMG 2 . . . Unknown 269,270 Aldolase HMG 2 . . . Unknown 271, 272 Aldolase HMG 2 . . . Unknown273, 274 Aldolase HMG 2 . . . Unknown 275, 276 Aldolase HMG 2 . . .Unknown 277, 278 Aldolase HMG 2 . . . Unknown 279, 280 Aldolase HMG 2 .. . Unknown 281, 282 Aldolase HMG 2 . . . Unknown 283, 284 Aldolase HMG2 . . . Unknown 285, 286 Aldolase HMG 2 . . . Unknown 287, 288 AldolaseHMG 2 . . . Unknown 289, 290 Aldolase HMG 2 . . . Unknown 291, 292Aldolase HMG 2 . . . Unknown 293, 294 Aldolase HMG 2 . . . Unknown 295,296 Aldolase HMG 2 . . . Unknown 297, 298 Aldolase HMG 2 . . . Unknown299, 300 Aldolase HMG 2.1 . . . Unknown 301, 302 Aldolase HMG 2.1 . . .Unknown 303, 304 Aldolase HMG 2.1 . . . Unknown 305, 306 Aldolase KHG4.1.2.14 Unknown 307, 308 Aldolase KHG 4.1.2.14 Unknown 309, 310Aldolase KHG 4.1.2.14 Unknown 311, 312 Aldolase KHG 4.1.2.14 Unknown313, 314 Aldolase KHG 4.1.2.14 Unknown 315, 316 Aldolase KHG 4.1.2.14Unknown 317, 318 Aldolase KHG 4.1.2.14 Unknown 319, 320 Aldolase KHG4.1.3.16 Unknown 321, 322 Aldolase KHG 4.1.2.14 Unknown 323, 324Aldolase KHG 4.1.2.14 Unknown 325, 326 Aldolase KHG 4.1.2.14 Unknown327, 328 Aldolase KHG 4.1.2.14 Unknown 329, 330 Aldolase KHG 4.1.3.16Unknown 331, 332 Aldolase KHG 4.1.2.14 Unknown 333, 334 Aldolase KHG4.1.2.14 Unknown

Polypeptides and peptides in accordance with the invention can beisolated from natural sources, be synthetic, or be recombinantlygenerated polypeptides. Peptides and proteins can be recombinantlyexpressed in vitro or in vivo. The peptides and polypeptides inaccordance with the invention can be made and isolated using any methodknown in the art. Polypeptide and peptides in accordance with theinvention can also be synthesized, in whole or in part, using chemicalmethods well known in the art. See such as Caruthers (1980) NucleicAcids Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser.225-232; Banga, A. K., Therapeutic Peptides and Proteins, Formulation,Processing and Delivery Systems (1995) Technomic Publishing Co.,Lancaster, Pa. For example, peptide synthesis can be performed usingvarious solid-phase techniques (see such as Roberge (1995) Science269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automatedsynthesis may be achieved, such as using the ABI 431A PeptideSynthesizer (Perkin Elmer) in accordance with the instructions providedby the manufacturer.

Peptides and polypeptides in accordance with the invention can also beglycosylated. The glycosylation can be added post-translationally eitherchemically or by cellular biosynthetic mechanisms, wherein the laterincorporates the use of known glycosylation motifs, which can be nativeto the sequence or can be added as a peptide or added in the nucleicacid coding sequence. The glycosylation can be O-linked or N-linked.

In some embodiments, when indicated, peptides and polypeptides inaccordance with the invention can include all “mimetic” and“peptidomimetic” forms. The terms “mimetic” and “peptidomimetic” referto a synthetic chemical compound which has substantially the samestructural and/or functional characteristics of the polypeptides inaccordance with the invention. The mimetic can be either entirelycomposed of synthetic, non-natural analogues of amino acids, or, is achimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The mimetic can also incorporate anyamount of natural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. As with polypeptides in accordance with the inventionwhich are conservative variants or members of a genus of polypeptides inaccordance with the invention (such as having about 50% or more sequenceidentity to a sequence in accordance with the invention), routineexperimentation will determine whether a mimetic is within the scope inaccordance with the invention, i.e., that its structure and/or functionis not substantially altered. Thus, in some embodiments, a mimeticcomposition is within the scope in accordance with the invention if ithas an aldolase, such as pyruvate aldolase, such as HMG and/or KHGaldolase enzyme, activity.

Polypeptide mimetic compositions in accordance with the invention cancontain any combination of non-natural structural components. In analternative embodiment, mimetic compositions in accordance with theinvention include one or all of the following three structural groups:a) residue linkage groups other than the natural amide bond (“peptidebond”) linkages; b) non-natural residues in place of naturally occurringamino acid residues; or c) residues which induce secondary structuralmimicry, i.e., to induce or stabilize a secondary structure, such as abeta turn, gamma turn, beta sheet, alpha helix conformation, and thelike. For example, a polypeptide in accordance with the invention can becharacterized as a mimetic when all or some of its residues are joinedby chemical means other than natural peptide bonds. Individualpeptidomimetic residues can be joined by peptide bonds, other chemicalbonds or coupling means, such as glutaraldehyde, N-hydroxysuccinimideesters, bifunctional maleimides, N,N′-dicyclohexylcarbodiimide (DCC) orN,N′-diisopropylcarbodiimide (DIC). Linking groups that can be analternative to the traditional amide bond (“peptide bond”) linkagesinclude, such as ketomethylene (such as —C(═O)—CH₂— for —C(═O)—NH—),aminomethylene (CH₂—NH), ethylene, olefin (CH═CH), ether (CH₂—O),thioether (CH₂—S), tetrazole (CN₄—), thiazole, retroamide, thioamide, orester (see Spatola (1983) in Chemistry and Biochemistry of Amino Acids,Peptides and Proteins, Vol. 7, pp 267-357, “Peptide BackboneModifications,” Marcell Dekker, NY).

A polypeptide in accordance with the invention can also be characterizedas a mimetic by containing all or some non-natural residues in place ofnaturally occurring amino acid residues. Non-natural residues are welldescribed in the scientific and patent literature; a few exemplarynon-natural compositions useful as mimetics of natural amino acidresidues and guidelines are described below. Mimetics of aromatic aminoacids can be generated by replacing by, such as D- or L-naphylalanine;D- or L-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2,3-, or4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; D- orL-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylainines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of anon-natural amino acid include, such as thiazolyl, thiophenyl,pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridylaromatic rings.

Mimetics of acidic amino acids can be generated by substitution by, suchas non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (such asaspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) such as1-cyclohexyl-3(2-morpholinyl-(4-ethyl)carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl)carbodiimide Aspartyl or glutamylcan also be converted to asparaginyl and glutaminyl residues by reactionwith ammonium ions. Mimetics of basic amino acids can be generated bysubstitution with, such as (in addition to lysine and arginine) theamino acids ornithine, citrulline, or (guanidino)-acetic acid, or(guanidino)alkyl-acetic acid, where alkyl is defined above. Nitrilederivative (such as containing the CN-moiety in place of COOH) can besubstituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, such as one or more conventional reagents, including, such asphenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin, insome embodiments under alkaline conditions. Tyrosine residue mimeticscan be generated by reacting tyrosyl with, such as aromatic diazoniumcompounds or tetranitromethane. N-acetylimidizol and tetranitromethanecan be used to form O-acetyl tyrosyl species and 3-nitro derivatives,respectively. Cysteine residue mimetics can be generated by reactingcysteinyl residues with, such as alpha-haloacetates such as2-chloroacetic acid or chloroacetamide and corresponding amines; to givecarboxymethyl or carboxyamidomethyl derivatives. Cysteine residuemimetics can also be generated by reacting cysteinyl residues with, suchas bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, such as succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, such as methionine sulfoxide.Mimetics of proline include, such as pipecolic acid, thiazolidinecarboxylic acid, 3- or 4-hydroxy proline, dehydroproline, 3- or4-methylproline, or 3,3,-dimethylproline. Histidine residue mimetics canbe generated by reacting histidyl with, such as diethylprocarbonate orpara-bromophenacyl bromide. Other mimetics include, such as thosegenerated by hydroxylation of proline and lysine; phosphorylation of thehydroxyl groups of seryl or threonyl residues; methylation of thealpha-amino groups of lysine, arginine and histidine; acetylation of theN-terminal amine; methylation of main chain amide residues orsubstitution with N-methyl amino acids; or amidation of C-terminalcarboxyl groups.

In some embodiments, a residue, such as an amino acid, of a polypeptidein accordance with the invention can also be replaced by an amino acid(or peptidomimetic residue) of the opposite chirality. In someembodiments, any amino acid naturally occurring in the L-configuration(which can also be referred to as the R or S, depending upon thestructure of the chemical entity) can be replaced with the amino acid ofthe same chemical structural type or a peptidomimetic, but of theopposite chirality, referred to as the D-amino acid, but also can bereferred to as the R- or S-form.

The invention also provides methods for modifying the polypeptides inaccordance with the invention by either natural processes, such aspost-translational processing (such as phosphorylation, acylation, etc),or by chemical modification techniques, and the resulting modifiedpolypeptides. Modifications can occur anywhere in the polypeptide,including the peptide backbone, the amino acid side-chains and the aminoor carboxyl termini. It will be appreciated that the same type ofmodification may be present in the same or varying degrees at severalsites in a given polypeptide. Also a given polypeptide may have manytypes of modifications. In some embodiments, modifications includeacetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of a phosphatidylinositol,cross-linking cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristolyation, oxidation, pegylation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,and transfer-RNA mediated addition of amino acids to protein such asarginylation. See, Creighton, T. E., Proteins—Structure and MolecularProperties 2nd Ed., W.H. Freeman and Company, New York (1993);Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York, pp. 1-12 (1983).

Solid-phase chemical peptide synthesis methods can also be used tosynthesize the polypeptide or fragments in accordance with theinvention. Such method have been known in the art since the early 1960's(Merrifield, R. B., J. Am. Chem. Soc., 85:2149-2154, 1963) (See alsoStewart, J. M. and Young, J. D., Solid Phase Peptide Synthesis, 2nd Ed.,Pierce Chemical Co., Rockford, Ill., pp. 11-12)) and have recently beenemployed in commercially available laboratory peptide design andsynthesis kits (Cambridge Research Biochemicals). Such commerciallyavailable laboratory kits have generally utilized the teachings of H. M.Geysen et al, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and providefor synthesizing peptides upon the tips of a multitude of “rods” or“pins” all of which are connected to a single plate. When such a systemis utilized, a plate of rods or pins is inverted and inserted into asecond plate of corresponding wells or reservoirs, which containsolutions for attaching or anchoring an appropriate amino acid to thepin's or rod's tips. By repeating such a process step, i.e., invertingand inserting the rod's and pin's tips into appropriate solutions, aminoacids are built into desired peptides. In addition, a number ofavailable FMOC peptide synthesis systems are available. For example,assembly of a polypeptide or fragment can be carried out on a solidsupport using an Applied Biosystems, Inc. Model 431A™ automated peptidesynthesizer. Such equipment provides ready access to the peptides inaccordance with the invention, either by direct synthesis or bysynthesis of a series of fragments that can be coupled using other knowntechniques.

The polypeptides in accordance with the invention include aldolase, suchas pyruvate aldolase, such as HMG and/or KHG aldolase enzymes in anactive or inactive form. For example, the polypeptides in accordancewith the invention include proproteins before “maturation” or processingof prepro sequences, such as by a proprotein-processing enzyme, such asa proprotein convertase to generate an “active” mature protein. Thepolypeptides in accordance with the invention include aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzymes inactive forother reasons, such as before “activation” by a post-translationalprocessing event, such as an endo- or exo-peptidase or proteinaseaction, a phosphorylation event, an amidation, a glycosylation or asulfation, a dimerization event, and the like. The polypeptides inaccordance with the invention include all active forms, including activesubsequences, such as catalytic domains or active sites, of the enzyme.

The invention includes immobilized aldolase, such as pyruvate aldolase,such as HMG and/or KHG aldolase enzymes, anti-aldolase, such asanti-pyruvate aldolase, such as anti-HMG and/or anti-KHG aldolaseantibodies and fragments thereof. In some embodiments, the inventionprovides methods for inhibiting aldolase, such as pyruvate aldolase,such as HMG and/or KHG aldolase enzyme, activity, such as using dominantnegative mutants or anti-aldolase, such as anti-pyruvate aldolase, suchas anti-HMG and/or anti-KHG aldolase antibodies in accordance with theinvention. In some embodiments, the invention includes heterocomplexes,such as fusion proteins, heterodimers, etc., comprising the aldolase,such as pyruvate aldolase, such as HMG and/or KHG aldolase enzymes inaccordance with the invention.

In some embodiments, polypeptides in accordance with the invention canhave an aldolase, such as pyruvate aldolase, such as HMG and/or KHGaldolase enzyme, activity under various conditions, such as at extremesin pH and/or temperature or, in some embodiments, in the presence ofoxidizing agents. In some embodiments, the invention provides methodsleading to alternative aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzyme preparations with different catalyticefficiencies and stabilities, such as towards temperature, oxidizingagents and changing wash conditions. In some embodiments, aldolase, suchas pyruvate aldolase, such as HMG and/or KHG aldolase enzyme variantscan be produced using techniques of site-directed mutagenesis and/orrandom mutagenesis. In some embodiments, directed evolution can be usedto produce a great variety of aldolase, such as pyruvate aldolase, suchas HMG and/or KHG aldolase enzyme variants with alternativespecificities and stability.

The proteins in accordance with the invention are also useful asresearch reagents to identify aldolase, such as pyruvate aldolase, suchas HMG and/or KHG aldolase enzyme modulators, such as activators orinhibitors of aldolase, such as pyruvate aldolase, such as HMG and/orKHG aldolase enzyme, activity. Briefly, test samples (compounds, broths,extracts, and the like) are added to aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzyme assays to determinetheir ability to inhibit substrate cleavage Inhibitors identified inthis way can be used in industry and research to reduce or preventundesired proteolysis. As with aldolase, such as pyruvate aldolase, suchas HMG and/or KHG aldolase enzymes, inhibitors can be combined toincrease the spectrum of activity.

The enzymes in accordance with the invention are also useful as researchreagents to digest proteins or in protein sequencing. For example, thealdolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzymes may be used to break polypeptides into smaller fragments forsequencing using, such as an automated sequencer.

The invention also provides methods of discovering new aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzymes using thenucleic acids, polypeptides and antibodies in accordance with theinvention. In some embodiments, phagemid libraries are screened forexpression-based discovery of aldolase, such as pyruvate aldolase, suchas HMG and/or KHG aldolase enzymes. In other embodiments, lambda phagelibraries are screened for expression-based discovery of aldolase, suchas pyruvate aldolase, such as HMG and/or KHG aldolase enzymes. Screeningof the phage or phagemid libraries can allow the detection of toxicclones; improved access to substrate; reduced need for engineering ahost, by-passing the potential for any bias resulting from mass excisionof the library; and, faster growth at low clone densities. Screening ofphage or phagemid libraries can be in liquid phase or in solid phase. Insome embodiments, the invention provides screening in liquid phase. Thisgives a greater flexibility in assay conditions; additional substrateflexibility; higher sensitivity for weak clones; and ease of automationover solid phase screening.

The invention provides screening methods using the proteins and nucleicacids in accordance with the invention and robotic automation to enablethe execution of many thousands of biocatalytic reactions and screeningassays in a short period of time, such as per day, as well as ensuring ahigh level of accuracy and reproducibility (see discussion of arrays,below). As a result, a library of derivative compounds can be producedin a matter of weeks. For further teachings on modification ofmolecules, including small molecules, see PCT/US94/09174; U.S. Pat. No.6,245,547.

In some embodiments, polypeptides or fragments in accordance with theinvention are obtained through biochemical enrichment or purificationprocedures. The sequence of potentially homologous polypeptides orfragments may be determined by aldolase, such as pyruvate aldolase, suchas HMG and/or KHG aldolase enzyme assays (see Examples 3, 4 and 5,below), gel electrophoresis and/or microsequencing. The sequence of theprospective polypeptide or fragment in accordance with the invention canbe compared to a polypeptide in accordance with the invention, or afragment, such as comprising at least about 5, 10, 15, 20, 25, 30, 35,40, 50, 75, 100, or 150 or more consecutive amino acids thereof usingany of the programs described above.

Another embodiment of the invention is an assay for identifyingfragments or variants in accordance with the invention, which retain theenzymatic function of the polypeptides in accordance with the invention.For example the fragments or variants of said polypeptides, may be usedto catalyze biochemical reactions, which indicate that the fragment orvariant retains the enzymatic activity of a polypeptide in accordancewith the invention. An exemplary assay for determining if fragments ofvariants retain the enzymatic activity of the polypeptides in accordancewith the invention includes the steps of: contacting the polypeptidefragment or variant with a substrate molecule under conditions whichallow the polypeptide fragment or variant to function and detectingeither a decrease in the level of substrate or an increase in the levelof the specific reaction product of the reaction between the polypeptideand substrate.

The present invention exploits the unique catalytic properties ofenzymes. Whereas the use of biocatalysts (i.e., purified or crudeenzymes, non-living or living cells) in chemical transformationsnormally requires the identification of a particular biocatalyst thatreacts with a specific starting compound, the present invention usesselected biocatalysts and reaction conditions that are specific forfunctional groups that are present in many starting compounds, such assmall molecules. Each biocatalyst is specific for one functional group,or several related functional groups and can react with many startingcompounds containing this functional group.

In some embodiments, the biocatalytic reactions produce a population ofderivatives from a single starting compound. These derivatives can besubjected to another round of biocatalytic reactions to produce a secondpopulation of derivative compounds. Thousands of variations of theoriginal small molecule or compound can be produced with each iterationof biocatalytic derivatization.

Enzymes react at specific sites of a starting compound without affectingthe rest of the molecule, a process which is very difficult to achieveusing traditional chemical methods. This high degree of biocatalyticspecificity provides the means to identify a single active compoundwithin the library. The library is characterized by the series ofbiocatalytic reactions used to produce it, a so-called “biosynthetichistory”. Screening the library for biological activities and tracingthe biosynthetic history identifies the specific reaction sequenceproducing the active compound. The reaction sequence is repeated and thestructure of the synthesized compound determined. This mode ofidentification, unlike other synthesis and screening approaches, doesnot require immobilization technologies and compounds can be synthesizedand tested free in solution using virtually any type of screening assay.It is important to note, that the high degree of specificity of enzymereactions on functional groups allows for the “tracking” of specificenzymatic reactions that make up the biocatalytically produced library.

In some embodiments, procedural steps are performed using roboticautomation enabling the execution of many thousands of biocatalyticreactions and/or screening assays per day as well as ensuring a highlevel of accuracy and reproducibility. Robotic automation can also beused to screen for aldolase activity to determine if a polypeptide iswithin the scope in accordance with the invention. As a result, in someembodiments, a library of derivative compounds can be produced in amatter of weeks which would take years to produce using “traditional”chemical or enzymatic screening methods.

In one embodiment, the invention provides methods for modifying smallmolecules, comprising contacting a polypeptide encoded by apolynucleotide described herein or enzymatically active fragmentsthereof with a small molecule to produce a modified small molecule. Alibrary of modified small molecules is tested to determine if a modifiedsmall molecule is present within the library, which exhibits a desiredactivity. A specific biocatalytic reaction which produces the modifiedsmall molecule of desired activity is identified by systematicallyeliminating each of the biocatalytic reactions used to produce a portionof the library and then testing the small molecules produced in theportion of the library for the presence or absence of the modified smallmolecule with the desired activity. The specific biocatalytic reactionswhich produce the modified small molecule of desired activity isoptionally repeated. The biocatalytic reactions are conducted with agroup of biocatalysts that react with distinct structural moieties foundwithin the structure of a small molecule, each biocatalyst is specificfor one structural moiety or a group of related structural moieties; andeach biocatalyst reacts with many different small molecules whichcontain the distinct structural moiety.

Aldolase, Such as Pyruvate Aldolase, Such as HMG and/or KHG AldolaseEnzyme Signal Sequences, Prepro and Catalytic Domains

The invention provides aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzyme signal sequences (such as signal peptides(SPs)), prepro domains and catalytic domains (CDs). The SPs, preprodomains and/or CDs in accordance with the invention can be isolated,synthetic or recombinant peptides or can be part of a fusion protein,such as a heterologous domain in a chimeric protein. In someembodiments, the invention provides nucleic acids encoding thesecatalytic domains (CDs), prepro domains and signal sequences (SPs, suchas a peptide having a sequence comprising/consisting of amino terminalresidues of a polypeptide in accordance with the invention).

The invention provides isolated, synthetic or recombinant signalsequences (such as signal peptides) consisting of or comprising asequence as set forth in residues 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 40, 1 to 41, 1 to 42, 1to 43, 1 to 44, 1 to 45, 1 to 46, or 1 to 47, or more, of a polypeptidein accordance with the invention, such as polypeptides in accordancewith the invention, see also Table 1, Examples 4, 5 and 6, below, andSequence Listing. For example, Table 1, above, sets forth exemplarysignal (leader) sequences in accordance with the invention, such as inthe polypeptide having a sequence as set forth in SEQ ID NO:66, encoded,such as by SEQ ID NO:65, has a signal sequence comprising (or consistingof) the amino terminal 27 residues, or, MSIVVTKIERAGAAAVAALRTSGVATV (SEQID NO:407) which corresponds to the first 27 amino acids of SEQ IDNO:66.

In some embodiments, the invention provides signal sequences comprisingthe first 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70 or more amino terminal residues of a polypeptidein accordance with the invention.

The invention includes polypeptides with or without a signal sequenceand/or a prepro sequence. In some embodiments, the invention includespolypeptides with heterologous signal sequences and/or prepro sequences.The prepro sequence (including a sequence in accordance with theinvention used as a heterologous prepro domain) can be located on theamino terminal or the carboxy terminal end of the protein. In someembodiments, the invention also includes isolated, synthetic orrecombinant signal sequences, prepro sequences and catalytic domains(such as “active sites”) comprising sequences in accordance with theinvention. The polypeptide comprising a signal sequence in accordancewith the invention can be an aldolase, such as pyruvate aldolase, HMGand/or KHG aldolase enzyme in accordance with the invention or anotheraldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme or another enzyme or other polypeptide. Methods for identifying“prepro” domain sequences and signal sequences are well known in theart, see Van de Ven (1993) Crit. Rev. Oncog. 4(2):115-136. For example,to identify a prepro sequence, the protein is purified from theextracellular space and the N-terminal protein sequence is determinedand compared to the unprocessed form.

The aldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme signal sequences (SPs) and/or prepro sequences in accordance withthe invention can be isolated, synthetic or recombinant peptides, or,sequences joined to another aldolase, such as pyruvate aldolase, such asHMG and/or KHG aldolase enzyme or a non-aldolase, such as non-pyruvatealdolase, e.g, non-HMG and/or non-KHG aldolase polypeptide, such as afusion (chimeric) protein. In some embodiments, the invention providespolypeptides comprising aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzyme signal sequences in accordance with theinvention. In some embodiments, polypeptides comprising aldolase, suchas pyruvate aldolase, such as HMG and/or KHG aldolase enzyme signalsequences SPs and/or prepro in accordance with the invention comprisesequences heterologous to an aldolase, such as pyruvate aldolase, HMGand/or KHG aldolase enzyme in accordance with the invention (such as afusion protein comprising an SP and/or prepro in accordance with theinvention and sequences from another aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzyme or a non-aldolase, suchas non-pyruvate aldolase, e.g, non-HMG and/or non-KHG aldolase protein).In some embodiments, the invention provides aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzymes in accordance with theinvention with heterologous SPs and/or prepro sequences, such assequences with a yeast signal sequence. An aldolase, such as pyruvatealdolase, HMG and/or KHG aldolase enzyme in accordance with theinvention can comprise a heterologous SP and/or prepro in a vector, suchas a pPIC series vector (Invitrogen, Carlsbad, Calif.).

In some embodiments, SPs and/or prepro sequences in accordance with theinvention are identified following identification of novel aldolase,such as pyruvate aldolase, such as HMG and/or KHG aldolase polypeptides.The pathways by which proteins are sorted and transported to theirproper cellular location are often referred to as protein targetingpathways. One of the most important elements in all of these targetingsystems is a short amino acid sequence at the amino terminus of a newlysynthesized polypeptide called the signal sequence. This signal sequencedirects a protein to its appropriate location in the cell and is removedduring transport or when the protein reaches its final destination. Mostlysosomal, membrane, or secreted proteins have an amino-terminal signalsequence that marks them for translocation into the lumen of theendoplasmic reticulum. The signal sequences can vary in length fromabout 10 to 65, or more, amino acid residues. Various methods ofrecognition of signal sequences are known to those of skill in the art.For example, in some embodiments, novel aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzyme signal peptides areidentified by a method referred to as SignalP. SignalP uses a combinedneural network which recognizes both signal peptides and their cleavagesites. (Nielsen (1997) “Identification of prokaryotic and eukaryoticsignal peptides and prediction of their cleavage sites.” ProteinEngineering 10:1-6.

In some embodiments, aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzymes in accordance with the invention do not haveSPs and/or prepro sequences or “domains.” In some embodiments, theinvention provides the aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzymes in accordance with the invention lacking allor part of an SP and/or a prepro domain. In some embodiments, theinvention provides nucleic acid sequences encoding a signal sequence(SP) and/or prepro from one aldolase, such as pyruvate aldolase, such asHMG and/or KHG aldolase enzyme operably linked to a nucleic acidsequence of a different aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzyme or, optionally, a signal sequence (SPs)and/or prepro domain from a non-aldolase, such as non-pyruvate aldolase,e.g, non-HMG and/or non-KHG aldolase protein may be desired.

The invention also provides isolated, synthetic or recombinantpolypeptides comprising signal sequences (SPs), prepro domain and/orcatalytic domains (CDs) in accordance with the invention andheterologous sequences. The heterologous sequences are sequences notnaturally associated (such as to a enzyme) with an SP, prepro domainand/or CD. The sequence to which the SP, prepro domain and/or CD are notnaturally associated can be on the SP's, prepro domain and/or CD's aminoterminal end, carboxy terminal end, and/or on both ends of the SP and/orCD. In some embodiments, the invention provides isolated, synthetic orrecombinant polypeptides comprising (or consisting of) a polypeptidecomprising a signal sequence (SP), prepro domain and/or catalytic domain(CD) in accordance with the invention with the proviso that it is notassociated with any sequence to which it is naturally associated (suchas an aldolase, such as pyruvate aldolase, HMG and/or KHG aldolaseenzyme sequence). Similarly, in some embodiments, the invention providesisolated, synthetic or recombinant nucleic acids encoding thesepolypeptides. Thus, in some embodiments, the isolated, synthetic orrecombinant nucleic acid in accordance with the invention comprisescoding sequence for a signal sequence (SP), prepro domain and/orcatalytic domain (CD) in accordance with the invention and aheterologous sequence (i.e., a sequence not naturally associated withthe a signal sequence (SP), prepro domain and/or catalytic domain (CD)in accordance with the invention). The heterologous sequence can be onthe 3′ terminal end, 5′ terminal end, and/or on both ends of the SP,prepro domain and/or CD coding sequence.

Hybrid (Chimeric) Aldolase, Such as Pyruvate Aldolase, Such as HMGand/or KHG Aldolase Enzymes and Peptide Libraries

In some embodiments, the invention provides hybrid aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzymes and fusionproteins, including peptide libraries, comprising sequences inaccordance with the invention. The peptide libraries in accordance withthe invention can be used to isolate peptide modulators (such asactivators or inhibitors) of targets, such as aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzyme substrates, receptors,enzymes. The peptide libraries in accordance with the invention can beused to identify formal binding partners of targets, such as ligands,such as cytokines, hormones and the like. In some embodiments, theinvention provides chimeric proteins comprising a signal sequence (SP),prepro domain and/or catalytic domain (CD) in accordance with theinvention or a combination thereof and a heterologous sequence (seeabove).

In some embodiments, the fusion proteins in accordance with theinvention (such as the peptide moiety) are conformationally stabilized(relative to linear peptides) to allow a higher binding affinity fortargets. In some embodiments, the invention provides fusions ofaldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzymes in accordance with the invention and other peptides, includingknown and random peptides. They can be fused in such a manner that thestructure of the aldolase, such as pyruvate aldolase, such as HMG and/orKHG aldolase enzymes is not significantly perturbed and the peptide ismetabolically or structurally conformationally stabilized. This allowsthe creation of a peptide library that is easily monitored both for itspresence within cells and its quantity.

Amino acid sequence variants in accordance with the invention can becharacterized by a predetermined nature of the variation, a feature thatsets them apart from a naturally occurring form, such as an allelic orinterspecies variation of an aldolase, such as pyruvate aldolase, HMGand/or KHG aldolase enzyme sequence. In some embodiments, the variantsin accordance with the invention exhibit the same qualitative biologicalactivity as the naturally occurring analogue. Alternatively, thevariants can be selected for having modified characteristics. In someembodiments, while the site or region for introducing an amino acidsequence variation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzyme variants screened forthe optimal combination of desired activity. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known, as discussed herein for example, M13 primermutagenesis and PCR mutagenesis. Screening of the mutants can be doneusing, such as assays of carbon-carbon bond formation or cleavage. Inother embodiments, amino acid substitutions can be single residues;insertions can be on the order of from about 1 to 20 amino acids,although considerably larger insertions can be done. Deletions can rangefrom about 1 to about 20, 30, 40, 50, 60, 70 residues or more. To obtaina final derivative with the optimal properties, substitutions,deletions, insertions or any combination thereof may be used. Generally,these changes are done on a few amino acids to minimize the alterationof the molecule. However, larger changes may be tolerated in certaincircumstances.

The invention provides aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzymes where the structure of the polypeptidebackbone, the secondary or the tertiary structure, such as analpha-helical or beta-sheet structure, has been modified. In someembodiments, the charge or hydrophobicity has been modified. In someembodiments, the bulk of a side chain has been modified. Substantialchanges in function or immunological identity are made by selectingsubstitutions that are less conservative. For example, substitutions canbe made which more significantly affect: the structure of thepolypeptide backbone in the area of the alteration, for example aalpha-helical or a beta-sheet structure; a charge or a hydrophobic siteof the molecule, which can be at an active site; or a side chain. Insome embodiments, the invention provides substitutions in polypeptide inaccordance with the invention where (a) a hydrophilic residues, such asseryl or threonyl, is substituted for (or by) a hydrophobic residue,such as leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteineor proline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, such as lysyl, arginyl, orhistidyl, is substituted for (or by) an electronegative residue, such asglutamyl or aspartyl; or (d) a residue having a bulky side chain, suchas phenylalanine, is substituted for (or by) one not having a sidechain, such as glycine. The variants can exhibit the same qualitativebiological activity (i.e., an aldolase, such as pyruvate aldolase, suchas HMG and/or KHG aldolase enzyme, activity) although variants can beselected to modify the characteristics of the aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzymes as needed.

In some embodiments, aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzymes in accordance with the invention compriseepitopes or purification tags, signal sequences or other fusionsequences, etc. In some embodiments, the aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzymes in accordance with theinvention can be fused to a random peptide to form a fusion polypeptide.By “fused” or “operably linked” herein is meant that the random peptideand the aldolase, such as pyruvate aldolase, such as HMG and/or KHGaldolase enzyme are linked together, in such a manner as to minimize thedisruption to the stability of the aldolase, such as pyruvate aldolase,such as HMG and/or KHG aldolase enzyme structure, such as it retainsaldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme, activity. The fusion polypeptide (or fusion polynucleotideencoding the fusion polypeptide) can comprise further components aswell, including multiple peptides at multiple loops.

In some embodiments, the peptides and nucleic acids encoding them arerandomized, either fully randomized or they are biased in theirrandomization, such as in nucleotide/residue frequency generally or perposition. “Randomized” means that each nucleic acid and peptide consistsof essentially random nucleotides and amino acids, respectively. In someembodiments, the nucleic acids which give rise to the peptides can bechemically synthesized, and thus may incorporate any nucleotide at anyposition. Thus, when the nucleic acids are expressed to form peptides,any amino acid residue may be incorporated at any position. Thesynthetic process can be designed to generate randomized nucleic acids,to allow the formation of all or most of the possible combinations overthe length of the nucleic acid, thus forming a library of randomizednucleic acids. The library can provide a sufficiently structurallydiverse population of randomized expression products to affect aprobabilistically sufficient range of cellular responses to provide oneor more cells exhibiting a desired response. Thus, the inventionprovides interaction libraries large enough so that at least one of itsmembers will have a structure that gives it affinity for some molecule,protein, or other factor.

In some embodiments, an aldolase, such as pyruvate aldolase, HMG and/orKHG aldolase enzyme in accordance with the invention is a multidomainenzyme that comprises a signal peptide, a carbohydrate binding module,an aldolase, such as pyruvate aldolase, HMG and/or KHG aldolase enzymecatalytic domain, a linker and/or another catalytic domain.

The invention provides methods and sequences for generating chimericpolypeptides which may encode biologically active hybrid polypeptides(such as hybrid aldolase, such as pyruvate aldolase, such as HMG and/orKHG aldolase enzymes). In some embodiments, the original polynucleotides(such as a nucleic acid in accordance with the invention) encodebiologically active polypeptides. In some embodiments, a method inaccordance with the invention produces new hybrid polypeptides byutilizing cellular processes which integrate the sequence of theoriginal polynucleotides such that the resulting hybrid polynucleotideencodes a polypeptide demonstrating activities derived, but different,from the original biologically active polypeptides (such as aldolase orantibody in accordance with the invention). For example, the originalpolynucleotides may encode a particular enzyme (such as aldolase) fromor found in different microorganisms. An enzyme encoded by a firstpolynucleotide from one organism or variant may, for example, functioneffectively under a particular environmental condition, such as highsalinity. An enzyme encoded by a second polynucleotide from a differentorganism or variant may function effectively under a differentenvironmental condition, such as extremely high temperatures. A hybridpolynucleotide containing sequences from the first and second originalpolynucleotides may encode an enzyme which exhibits characteristics ofboth enzymes encoded by the original polynucleotides. Thus, the enzymeencoded by the hybrid polynucleotide in accordance with the inventionmay function effectively under environmental conditions shared by eachof the enzymes encoded by the first and second polynucleotides, such ashigh salinity and extreme temperatures.

In some embodiments, a hybrid polypeptide generated by a method inaccordance with the invention may exhibit specialized enzyme activitynot displayed in the original enzymes. For example, followingrecombination and/or reductive reassortment of polynucleotides encodingaldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzymes, the resulting hybrid polypeptide encoded by a hybridpolynucleotide can be screened for specialized non-aldolase, such asnon-pyruvate aldolase, such as non-HMG and/or non-KHG-aldolase enzymeactivities, such as hydrolase, peptidase, phosphorylase, etc.,activities, obtained from each of the original enzymes. In someembodiments, the hybrid polypeptide is screened to ascertain thosechemical functionalities which distinguish the hybrid polypeptide fromthe original parent polypeptides, such as the temperature, pH or saltconcentration at which the hybrid polypeptide functions.

In some embodiments, the invention relates to a method for producing abiologically active hybrid polypeptide and screening such a polypeptidefor enhanced activity by:

-   1) introducing at least a first polynucleotide in operable linkage    and a second polynucleotide in operable linkage, the at least first    polynucleotide and second polynucleotide sharing at least one region    of partial sequence homology, into a suitable host cell;-   2) growing the host cell under conditions which promote sequence    reorganization resulting in a hybrid polynucleotide in operable    linkage;-   3) expressing a hybrid polypeptide encoded by the hybrid    polynucleotide;-   4) screening the hybrid polypeptide under conditions which promote    identification of enhanced biological activity; and-   5) isolating the a polynucleotide encoding the hybrid polypeptide.

Isolating and Discovering Aldolase Enzymes

The invention provides methods for isolating and discovering aldolase,such as pyruvate aldolase, HMG and/or KHG aldolase enzymes and thenucleic acids that encode them. Polynucleotides or enzymes may beisolated from individual organisms (“isolates”), collections oforganisms that have been grown in defined media (“enrichment cultures”),or, uncultivated organisms (“environmental samples”). The organisms canbe isolated by, such as in vivo biopanning (see discussion, below). Theuse of a culture-independent approach to derive polynucleotides encodingnovel bioactivities from environmental samples is most preferablebecause it allows one to access untapped resources of biodiversity.Polynucleotides or enzymes also can be isolated from any one of numerousorganisms, such as bacteria. In addition to whole cells, polynucleotidesor enzymes also can be isolated from crude enzyme preparations derivedfrom cultures of these organisms, such as bacteria.

“Environmental libraries” are generated from environmental samples andrepresent the collective genomes of naturally occurring organismsarchived in cloning vectors that can be propagated in suitableprokaryotic hosts. Because the cloned DNA is initially extracteddirectly from environmental samples, the libraries are not limited tothe small fraction of prokaryotes that can be grown in pure culture.Additionally, a normalization of the environmental DNA present in thesesamples could allow more equal representation of the DNA from all of thespecies present in the original sample. This can dramatically increasethe efficiency of finding interesting genes from minor constituents ofthe sample which may be under-represented by several orders of magnitudecompared to the dominant species.

In some embodiments, gene libraries generated from one or moreuncultivated microorganisms are screened for an activity of interest.Potential pathways encoding bioactive molecules of interest are firstcaptured in prokaryotic cells in the form of gene expression libraries.In some embodiments, polynucleotides encoding activities of interest areisolated from such libraries and introduced into a host cell. The hostcell is grown under conditions which promote recombination and/orreductive reassortment creating potentially active biomolecules withnovel or enhanced activities.

In vivo biopanning may be performed utilizing a FACS-based andnon-optical (such as magnetic) based machines. In some embodiments,complex gene libraries are constructed with vectors which containelements which stabilize transcribed RNA. For example, the inclusion ofsequences which result in secondary structures such as hairpins whichare designed to flank the transcribed regions of the RNA would serve toenhance their stability, thus increasing their half life within thecell. The probe molecules used in the biopanning process consist ofoligonucleotides labeled with reporter molecules that only fluoresceupon binding of the probe to a target molecule. These probes areintroduced into the recombinant cells from the library using one ofseveral transformation methods. The probe molecules bind to thetranscribed target mRNA resulting in DNA/RNA heteroduplex molecules.Binding of the probe to a target will yield a fluorescent signal whichis detected and sorted by the FACS machine during the screening process.

In some embodiments, subcloning is performed to further isolatesequences of interest. In subcloning, a portion of DNA is amplified,digested, generally by restriction enzymes, to cut out the desiredsequence, the desired sequence is ligated into a recipient vector and isamplified. At each step in subcloning, the portion is examined for theactivity of interest, in order to ensure that DNA that encodes thestructural protein has not been excluded. The insert may be purified atany step of the subcloning, for example, by gel electrophoresis prior toligation into a vector or where cells containing the recipient vectorand cells not containing the recipient vector are placed on selectivemedia containing, for example, an antibiotic, which will kill the cellsnot containing the recipient vector. Specific methods of subcloning cDNAinserts into vectors are well-known in the art (Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press (1989)). In other embodiments, the enzymes inaccordance with the invention are subclones. Such subclones may differfrom the parent clone by, for example, length, a mutation, a tag or alabel.

The microorganisms from which the polynucleotide may be discovered,isolated or prepared include prokaryotic microorganisms, such asEubacteria and Archaebacteria and lower eukaryotic microorganisms suchas fungi, some algae and protozoa. Polynucleotides may be discovered,isolated or prepared from environmental samples in which case thenucleic acid may be recovered without culturing of an organism orrecovered from one or more cultured organisms. In some embodiments, suchmicroorganisms may be extremophiles, such as hyperthermophiles,psychrophiles, psychrotrophs, halophiles, barophiles and acidophiles.Polynucleotides encoding enzymes isolated from extremophilicmicroorganisms can be used. Enzymes of this invention can function attemperatures above 100° C., such as those found in terrestrial hotsprings and deep sea thermal vents, or at temperatures below 0° C., suchas those found in arctic waters, in a saturated salt environment, suchas those found in the Dead Sea, at pH values around 0, such as thosefound in coal deposits and geothermal sulfur-rich springs, or at pHvalues greater than 11, such as those found in sewage sludge. In someembodiments, enzymes in accordance with the invention have high activitythroughout a wide range of temperatures and pHs.

Polynucleotides selected and isolated as hereinabove described areintroduced into a suitable host cell. A suitable host cell is any cellwhich is capable of promoting recombination and/or reductivereassortment. The selected polynucleotides are, in some embodiments,already in a vector which includes appropriate control sequences. Thehost cell can be a higher eukaryotic cell, such as a mammalian cell, ora lower eukaryotic cell, such as a yeast cell, or, in some embodiments,the host cell can be a prokaryotic cell, such as a bacterial cell.Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-Dextran mediated transfection, orelectroporation.

Exemplary hosts include bacterial cells, such as E. coli, Streptomyces,Salmonella typhimurium; fungal cells, such as yeast; insect cells suchas Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS orBowes melanoma; adenoviruses; and plant cells; see discussion, above.The selection of an appropriate host is deemed to be within the scope ofthose skilled in the art from the teachings herein.

Various mammalian cell culture systems can be employed to expressrecombinant protein; examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described in“SV40-transformed simian cells support the replication of early SV40mutants” (Gluzman, 1981) and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors can comprise an origin ofreplication, a suitable promoter and enhancer and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

In other embodiments, nucleic acids, polypeptides and methods inaccordance with the invention are used in biochemical pathways, or togenerate novel polynucleotides encoding biochemical pathways from one ormore operons or gene clusters or portions thereof. For example, bacteriaand many eukaryotes have a coordinated mechanism for regulating geneswhose products are involved in related processes. The genes areclustered, in structures referred to as “gene clusters,” on a singlechromosome and are transcribed together under the control of a singleregulatory sequence, including a single promoter which initiatestranscription of the entire cluster. Thus, a gene cluster is a group ofadjacent genes that are either identical or related, usually as to theirfunction (an example of a biochemical pathway encoded by gene clustersare polyketides).

In some embodiments, gene cluster DNA is isolated from differentorganisms and ligated into vectors, such as vectors containingexpression regulatory sequences which can control and regulate theproduction of a detectable protein or protein-related array activityfrom the ligated gene clusters. Use of vectors which have anexceptionally large capacity for exogenous DNA introduction can beappropriate for use with such gene clusters and are described by way ofexample herein to include the f-factor (or fertility factor) of E. coli.This f-factor of E. coli is a plasmid which affects high-frequencytransfer of itself during conjugation and is ideal to achieve and stablypropagate large DNA fragments, such as gene clusters from mixedmicrobial samples. In one embodiment, cloning vectors, referred to as“fosmids” or bacterial artificial chromosome (BAC) vectors are used.These are derived from E. coli f-factor which is able to stablyintegrate large segments of genomic DNA. When integrated with DNA from amixed uncultured environmental sample, this makes it possible to achievelarge genomic fragments in the form of a stable “environmental DNAlibrary.” Another type of vector for use in the present invention is acosmid vector. Cosmid vectors were originally designed to clone andpropagate large segments of genomic DNA. Cloning into cosmid vectors isdescribed in detail in Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989). Onceligated into an appropriate vector, two or more vectors containingdifferent polyketide synthase gene clusters can be introduced into asuitable host cell. Regions of partial sequence homology shared by thegene clusters will promote processes which result in sequencereorganization resulting in a hybrid gene cluster. The novel hybrid genecluster can then be screened for enhanced activities not found in theoriginal gene clusters.

Methods for screening for various enzyme activities are known to thoseof skill in the art and are discussed throughout the presentspecification, see Examples 1, 2 and 3, below. Such methods may beemployed when isolating the polypeptides and polynucleotides inaccordance with the invention.

In some embodiments, the invention provides methods for discovering andisolating aldolases, such as pyruvate aldolase, such as HMG and/or KHGaldolase, or compounds to modify the activity of these enzymes, using awhole cell approach (see discussion, below). Putative clones encodingaldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolasefrom genomic DNA library can be screened.

Screening Methodologies and “On-Line” Monitoring Devices

In practicing the methods in accordance with the invention, a variety ofapparatus and methodologies can be used to in conjunction with thepolypeptides and nucleic acids in accordance with the invention, such asto screen polypeptides for aldolase, such as pyruvate aldolase, such asHMG and/or KHG aldolase enzyme, activity, to screen compounds aspotential modulators, such as activators or inhibitors, of an aldolase,such as pyruvate aldolase, such as HMG and/or KHG aldolase enzyme,activity, for antibodies that bind to a polypeptide in accordance withthe invention, for nucleic acids that hybridize to a nucleic acid inaccordance with the invention, to screen for cells expressing apolypeptide in accordance with the invention and the like. In additionto the array formats described in detail below for screening samples,alternative formats can also be used to practice the methods inaccordance with the invention. Such formats include, for example, massspectrometers, chromatographs, such as high-throughput HPLC and otherforms of liquid chromatography, and smaller formats, such as 1536-wellplates, 384-well plates and so on. High throughput screening apparatuscan be adapted and used to practice the methods in accordance with theinvention, see U.S. Patent Application Nos. 20020001809; 20050272044.

Capillary Arrays

Nucleic acids or polypeptides in accordance with the invention can beimmobilized to or applied to an array. Arrays can be used to screen foror monitor libraries of compositions (such as small molecules,antibodies, nucleic acids, etc.) for their ability to bind to ormodulate the activity of a nucleic acid or a polypeptide in accordancewith the invention. Capillary arrays, such as the GIGAMATRIX™, DiversaCorporation, San Diego, Calif.; and arrays described in, such as U.S.Patent Application No. 20020080350 A1; WO 0231203 A; WO 0244336 A,provide an alternative apparatus for holding and screening samples. Insome embodiments, the capillary array includes a plurality ofcapillaries formed into an array of adjacent capillaries, wherein eachcapillary comprises at least one wall defining a lumen for retaining asample. The lumen may be cylindrical, square, hexagonal or any othergeometric shape so long as the walls form a lumen for retention of aliquid or sample. The capillaries of the capillary array can be heldtogether in close proximity to form a planar structure. The capillariescan be bound together, by being fused (such as where the capillaries aremade of glass), glued, bonded, or clamped side-by-side. Additionally,the capillary array can include interstitial material disposed betweenadjacent capillaries in the array, thereby forming a solid planar devicecontaining a plurality of through-holes.

A capillary array can be formed of any number of individual capillaries,for example, a range from 100 to 4,000,000 capillaries. Further, acapillary array having about 100,000 or more individual capillaries canbe formed into the standard size and shape of a Microtiter® plate forfitment into standard laboratory equipment. The lumens are filledmanually or automatically using either capillary action ormicroinjection using a thin needle. Samples of interest may subsequentlybe removed from individual capillaries for further analysis orcharacterization. For example, a thin, needle-like probe is positionedin fluid communication with a selected capillary to either add orwithdraw material from the lumen.

In a single-pot screening assay, the assay components are mixed yieldinga solution of interest, prior to insertion into the capillary array. Thelumen is filled by capillary action when at least a portion of the arrayis immersed into a solution of interest. Chemical or biologicalreactions and/or activity in each capillary are monitored for detectableevents. A detectable event is often referred to as a “hit”, which canusually be distinguished from “non-hit” producing capillaries by opticaldetection. Thus, capillary arrays allow for massively parallel detectionof “hits”.

In a multi-pot screening assay, a polypeptide or nucleic acid, such as aligand, can be introduced into a first component, which is introducedinto at least a portion of a capillary of a capillary array. An airbubble can then be introduced into the capillary behind the firstcomponent. A second component can then be introduced into the capillary,wherein the second component is separated from the first component bythe air bubble. The first and second components can then be mixed byapplying hydrostatic pressure to both sides of the capillary array tocollapse the bubble. The capillary array is then monitored for adetectable event resulting from reaction or non-reaction of the twocomponents.

In a binding screening assay, a sample of interest can be introduced asa first liquid labeled with a detectable particle into a capillary of acapillary array, wherein the lumen of the capillary is coated with abinding material for binding the detectable particle to the lumen. Thefirst liquid may then be removed from the capillary tube, wherein thebound detectable particle is maintained within the capillary, and asecond liquid may be introduced into the capillary tube. The capillaryis then monitored for a detectable event resulting from reaction ornon-reaction of the particle with the second liquid.

Arrays, or “Biochips”

Nucleic acids or polypeptides in accordance with the invention can beimmobilized to or applied to an array. Arrays can be used to screen foror monitor libraries of compositions (such as small molecules,antibodies, nucleic acids, etc.) for their ability to bind to ormodulate the activity of a nucleic acid or a polypeptide in accordancewith the invention. For example, in some embodiments of the invention, amonitored parameter is transcript expression of an aldolase, such aspyruvate aldolase, HMG and/or KHG aldolase enzyme gene. One or more, or,all the transcripts of a cell can be measured by hybridization of asample comprising transcripts of the cell, or, nucleic acidsrepresentative of or complementary to transcripts of a cell, byhybridization to immobilized nucleic acids on an array, or “biochip.” Byusing an “array” of nucleic acids on a microchip, some or all of thetranscripts of a cell can be simultaneously quantified. Alternatively,arrays comprising genomic nucleic acid can also be used to determine thegenotype of a newly engineered strain made by the methods in accordancewith the invention. Polypeptide arrays” can also be used tosimultaneously quantify a plurality of proteins. The present inventioncan be practiced with any known “array,” also referred to as a“microarray” or “nucleic acid array” or “polypeptide array” or “antibodyarray” or “biochip,” or variation thereof. Arrays are generically aplurality of “spots” or “target elements,” each target elementcomprising a defined amount of one or more biological molecules, such asoligonucleotides, immobilized onto a defined area of a substrate surfacefor specific binding to a sample molecule, such as mRNA transcripts.

The terms “array” or “microarray” or “biochip” or “chip” as used hereinis a plurality of target elements, each target element comprising adefined amount of one or more polypeptides (including antibodies) ornucleic acids immobilized onto a defined area of a substrate surface, asdiscussed in further detail, below.

In practicing the methods in accordance with the invention, any knownarray and/or method of making and using arrays can be incorporated inwhole or in part, or variations thereof, as described, for example, inU.S. Pat. Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270;6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098;5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854;5,807,522; 5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; seealso, such as WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; seealso, such as Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997)Biotechniques 23:1087-1092; Kern (1997) Biotechniques 23:120-124;Solinas-Toldo (1997) Genes, Chromosomes & Cancer 20:399-407; Bowtell(1999) Nature Genetics Supp. 21:25-32. See also published U.S. patentapplications Nos. 20010018642; 20010019827; 20010016322; 20010014449;20010014448; 20010012537; 20010008765.

Antibodies and Antibody-Based Screening Methods

The invention provides isolated, synthetic or recombinant antibodiesthat specifically bind to an aldolase, such as pyruvate aldolase, HMGand/or KHG aldolase enzyme in accordance with the invention. Theseantibodies can be used to isolate, identify or quantify the aldolase,such as pyruvate aldolase, such as HMG and/or KHG aldolase enzymes inaccordance with the invention or related polypeptides. These antibodiescan be used to isolate other polypeptides within the scope the inventionor other related aldolase, such as pyruvate aldolase, such as HMG and/orKHG aldolase enzymes. The antibodies can be designed to bind to anactive site of an aldolase, such as pyruvate aldolase, HMG and/or KHGaldolase enzyme. Thus, the invention provides methods of inhibitingaldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzymes using the antibodies in accordance with the invention (seediscussion above regarding applications for anti-aldolase, such asanti-pyruvate aldolase, such as anti-HMG and/or anti-KHG aldolase enzymecompositions in accordance with the invention).

The term “antibody” includes a peptide or polypeptide derived from,modeled after or substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof, capable of specificallybinding an antigen or epitope, see Fundamental Immunology, ThirdEdition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J.Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys.Methods 25:85-97. The term antibody includes antigen-binding portions,i.e., “antigen binding sites,” (such as fragments, subsequences,complementarity determining regions (CDRs)) that retain capacity to bindantigen, including (i) a Fab fragment, a monovalent fragment consistingof the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Single chain antibodies arealso included by reference in the term “antibody.”

The invention provides fragments of the enzymes in accordance with theinvention (such as peptides) including immunogenic fragments (such assubsequences) of a polypeptide in accordance with the invention. In someembodiments, the invention provides compositions comprising apolypeptide or peptide in accordance with the invention and adjuvants orcarriers and the like.

The antibodies can be used in immunoprecipitation, staining,immunoaffinity columns, and the like. If desired, nucleic acid sequencesencoding for specific antigens can be generated by immunization followedby isolation of polypeptide or nucleic acid, amplification or cloningand immobilization of polypeptide onto an array in accordance with theinvention. Alternatively, the methods in accordance with the inventioncan be used to modify the structure of an antibody produced by a cell tobe modified, such as an antibody's affinity can be increased ordecreased. Furthermore, the ability to make or modify antibodies can bea phenotype engineered into a cell by the methods in accordance with theinvention.

Methods of immunization, producing and isolating antibodies (polyclonaland monoclonal) are known to those of skill in the art and described inthe scientific and patent literature, see Coligan, CURRENT PROTOCOLS INIMMUNOLOGY, Wiley/Greene, N.Y. (1991); Stites (eds.) BASIC AND CLINICALIMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos, Calif.(“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2ded.) Academic Press, New York, N.Y. (1986); Kohler (1975) Nature256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, Cold SpringHarbor Publications, New York. Antibodies also can be generated invitro, such as using recombinant antibody binding site expressing phagedisplay libraries, in addition to the traditional in vivo methods usinganimals. See, Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz (1997)Annu. Rev. Biophys. Biomol. Struct. 26:27-45.

The polypeptides in accordance with the invention or fragmentscomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof, may also be used to generate antibodieswhich bind specifically to the polypeptides or fragments. The resultingantibodies may be used in immunoaffinity chromatography procedures toisolate or purify the polypeptide or to determine whether thepolypeptide is present in a biological sample. In such procedures, aprotein preparation, such as an extract, or a biological sample iscontacted with an antibody capable of specifically binding to one of thepolypeptides in accordance with the invention, or fragments comprisingat least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutiveamino acids thereof.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to one of the polypeptides in accordancewith the invention, or fragment thereof. After a wash to removenon-specifically bound proteins, the specifically bound polypeptides areeluted.

The ability of proteins in a biological sample to bind to the antibodymay be determined using any of a variety of procedures familiar to thoseskilled in the art. For example, binding may be determined by labelingthe antibody with a detectable label such as a fluorescent agent, anenzymatic label, or a radioisotope. Alternatively, binding of theantibody to the sample may be detected using a secondary antibody havingsuch a detectable label thereon. Particular assays include ELISA assays,sandwich assays, radioimmunoassays and Western Blots.

Polyclonal antibodies generated against the polypeptides in accordancewith the invention, or fragments comprising at least 5, 10, 15, 20, 25,30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof can beobtained by direct injection of the polypeptides into an animal or byadministering the polypeptides to an animal, for example, a nonhuman.The antibody so obtained can bind the polypeptide itself. In thismanner, even a sequence encoding only a fragment of the polypeptide canbe used to generate antibodies which may bind to the whole nativepolypeptide. Such antibodies can then be used to isolate the polypeptidefrom cells expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, Nature,256:495-497, 1975), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., Immunology Today 4:72, 1983) and theEBV-hybridoma technique (Cole, et al., 1985, in Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies tothe polypeptides in accordance with the invention, or fragmentscomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof. Alternatively, transgenic mice may beused to express humanized antibodies to these polypeptides or fragmentsthereof.

Antibodies generated against the polypeptides in accordance with theinvention, or fragments comprising at least 5, 10, 15, 20, 25, 30, 35,40, 50, 75, 100, or 150 consecutive amino acids thereof may be used inscreening for similar polypeptides from other organisms and samples. Insuch techniques, polypeptides from the organism are contacted with theantibody and those polypeptides which specifically bind the antibody aredetected. Any of the procedures described above may be used to detectantibody binding. One such screening assay is described in Shulman H,Eberhard A, Eberhard C, Ulitzur S, Keinan E, Bioorg Med Chem. Lett. 2000Oct. 16; 10(20):2353-6, Highly sensitive and rapid detection of antibodycatalysis by luminescent bacteria.

Kits

The invention provides kits comprising the compositions, such as nucleicacids, expression cassettes, vectors, cells, transgenic seeds or plantsor plant parts, polypeptides (such as an aldolase enzyme) and/orantibodies in accordance with the invention. The kits also can containinstructional material teaching the methodologies and industrial,medical and dietary uses in accordance with the invention, as describedherein.

Whole Cell Engineering and Measuring Metabolic Parameters

The methods in accordance with the invention provide whole cellevolution, or whole cell engineering, of a cell to develop a new cellstrain having a new phenotype, such as a new or modified aldolase, suchas pyruvate aldolase, such as HMG and/or KHG aldolase enzyme, activity,by modifying the genetic composition of the cell. See U.S. patentapplication no. 20040033975.

The genetic composition can be modified by addition to the cell of anucleic acid in accordance with the invention, such as a coding sequencefor an enzyme in accordance with the invention. See WO0229032;WO0196551.

To detect the new phenotype, at least one metabolic parameter of amodified cell is monitored in the cell in a “real time” or “on-line”time frame. In some embodiments, a plurality of cells, such as a cellculture, is monitored in “real time” or “on-line.” In some embodiments,a plurality of metabolic parameters is monitored in “real time” or“on-line.” Metabolic parameters can be monitored using the aldolase,such as pyruvate aldolase, such as HMG and/or KHG aldolase enzymes inaccordance with the invention.

Metabolic flux analysis (MFA) is based on a known biochemistryframework. A linearly independent metabolic matrix is constructed basedon the law of mass conservation and on the pseudo-steady statehypothesis (PSSH) on the intracellular metabolites. In practicing themethods in accordance with the invention, metabolic networks areestablished, including the:

-   -   identity of all pathway substrates, products and intermediary        metabolites    -   identity of all the chemical reactions interconverting the        pathway metabolites, the stoichiometry of the pathway reactions,    -   identity of all the enzymes catalyzing the reactions, the enzyme        reaction kinetics,    -   the regulatory interactions between pathway components, such as        allosteric interactions, enzyme-enzyme interactions etc,    -   intracellular compartmentalization of enzymes or any other        supramolecular organization of the enzymes, and,    -   the presence of any concentration gradients of metabolites,        enzymes or effector molecules or diffusion barriers to their        movement.

Once the metabolic network for a given strain is built, mathematicpresentation by matrix notion can be introduced to estimate theintracellular metabolic fluxes if the on-line metabolome data isavailable. Metabolic phenotype relies on the changes of the wholemetabolic network within a cell. Metabolic phenotype relies on thechange of pathway utilization with respect to environmental conditions,genetic regulation, developmental state and the genotype, etc. In someembodiments of the methods in accordance with the invention, after theon-line MFA calculation, the dynamic behavior of the cells, theirphenotype and other properties are analyzed by investigating the pathwayutilization. For example, if the glucose supply is increased and theoxygen decreased during the yeast fermentation, the utilization ofrespiratory pathways will be reduced and/or stopped, and the utilizationof the fermentative pathways will dominate. Control of physiologicalstate of cell cultures will become possible after the pathway analysis.The methods in accordance with the invention can help determine how tomanipulate the fermentation by determining how to change the substratesupply, temperature, use of inducers, etc. to control the physiologicalstate of cells to move along desirable direction. In practicing themethods in accordance with the invention, the MFA results can also becompared with transcriptome and proteome data to design experiments andprotocols for metabolic engineering or gene shuffling, etc.

In practicing the methods in accordance with the invention, any modifiedor new phenotype can be conferred and detected, including new orimproved characteristics in the cell. Any aspect of metabolism or growthcan be monitored.

Monitoring Expression of an mRNA Transcript

In some embodiments of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of an mRNA transcript (such asan aldolase, such as pyruvate aldolase, HMG and/or KHG aldolase enzymemessage) or generating new (such as aldolase, such as pyruvate aldolase,such as HMG and/or KHG aldolase enzyme) transcripts in a cell. Thisincreased or decreased expression can be traced by testing for thepresence of an aldolase, such as pyruvate aldolase, HMG and/or KHGaldolase enzyme in accordance with the invention or by aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzyme activityassays. mRNA transcripts, or messages, also can be detected andquantified by any method known in the art, including, such as Northernblots, quantitative amplification reactions, hybridization to arrays,and the like. Quantitative amplification reactions include, such asquantitative PCR, including, such as quantitative reverse transcriptionpolymerase chain reaction, or RT-PCR; quantitative real time RT-PCR, or“real-time kinetic RT-PCR” (see Kreuzer (2001) Br. J. Haematol.114:313-318; Xia (2001) Transplantation 72:907-914).

In some embodiments of the invention, the engineered phenotype isgenerated by knocking out expression of a homologous gene. The gene'scoding sequence or one or more transcriptional control elements can beknocked out, such as promoters or enhancers. Thus, the expression of atranscript can be completely ablated or only decreased.

In some embodiments of the invention, the engineered phenotype comprisesincreasing the expression of a homologous gene. This can be effected byknocking out of a negative control element, including a transcriptionalregulatory element acting in cis- or trans-, or, mutagenizing a positivecontrol element. One or more, or, all the transcripts of a cell can bemeasured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array.

Monitoring Expression of a Polypeptides, Peptides and Amino Acids

In some embodiments of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of a polypeptide (such as analdolase, such as pyruvate aldolase, HMG and/or KHG aldolase enzyme) orgenerating new polypeptides in a cell. This increased or decreasedexpression can be traced by determining the amount of aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzyme present or byaldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzyme activity assays. Polypeptides, peptides and amino acids also canbe detected and quantified by any method known in the art, including,such as nuclear magnetic resonance (NMR), spectrophotometry, radiography(protein radiolabeling), electrophoresis, capillary electrophoresis,high performance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, various immunological methods,such as immunoprecipitation, immunodiffusion, immuno-electrophoresis,radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs),immuno-fluorescent assays, gel electrophoresis (such as SDS-PAGE),staining with antibodies, fluorescent activated cell sorter (FACS),pyrolysis mass spectrometry, Fourier-Transform Infrared Spectrometry,Raman spectrometry, GC-MS, and LC-Electrospray andcap-LC-tandem-electrospray mass spectrometries, and the like. Novelbioactivities can also be screened using methods, or variations thereof,described in U.S. Pat. No. 6,057,103. Furthermore, as discussed below indetail, one or more, or, all the polypeptides of a cell can be measuredusing a protein array.

Industrial, Pharmaceutical and Other Applications

Polypeptides in accordance with the invention (such as having aldolase,such as pyruvate aldolase, such as HMG and/or KHG aldolase) can catalyzethe formation or cleavage of carbon-carbon bonds. The enzymes inaccordance with the invention can be highly selective catalysts. In someembodiments, the invention provides industrial processes using enzymesin accordance with the invention, such as in the pharmaceutical ornutrient (diet) supplement industry, in the food and feed industries,such as in methods for making food and feed products and food and feedadditives. In some embodiments, the invention provides processes usingenzymes in accordance with the invention in the medical industry, suchas to make pharmaceuticals or dietary aids or supplements, or foodsupplements and additives.

Biomass Conversion and Production of Clean Bio Fuels

The invention provides enzymes, such aldolases, including pyruvatealdolases such as, without limitation, HMG and/or KHG aldolases(including mixtures, or “cocktails” of enzymes) and methods for theconversion of a biomass or any lignocellulosic material (e.g., anycomposition comprising cellulose, hemicellulose and lignin), to fuels(e.g., bioethanol, biobutanol, biopropanol, biomethanol, biodiesel),using the enzymes of the invention, in addition to feeds, foods andchemicals. Thus, the compositions and methods of the invention provideeffective and sustainable alternatives or adjuncts to use ofpetroleum-based products, e.g., as a mixture of bioethanol and gasoline.The invention provides organisms expressing enzymes of the invention forparticipation in chemical cycles involving natural biomass conversion.In one embodiment, enzymes and methods for the conversion are used inenzyme ensembles for the efficient depolymerization of cellulosic andhemicellulosic polymers to metabolizeable carbon moieties. The inventionprovides methods for discovering and implementing the most effective ofenzymes to enable these important new “biomass conversion” andalternative energy industrial processes.

The methods of the invention also include taking the convertedlignocellulosic material (processed by enzymes of the invention) andmaking it into a fuel (e.g. bioethanol, biobutanol, biopropanol,biomethanol, biodiesel) by fermentation and/or by chemical synthesis. Inone embodiment, the produced sugars are fermented and/or thenon-fermentable products are gasified.

The enzymes of the invention (including, for example, organisms, such asmicroorganisms, e.g., fungi, yeast or bacteria, making and in someembodiments secreting recombinant enzymes of the invention) can be usedin or included/integrated at any stage of any biomass conversionprocess, e.g., at any one step, several steps, or included in all of thesteps, or all of the following methods of biomass conversion processes,or all of these biofuel alternatives:

Direct combustion: the burning of material by direct heat and is thesimplest biomass technology; can be very economical if a biomass sourceis nearby.

Pyrolysis: is the thermal degradation of biomass by heat in the absenceof oxygen. In one embodiment, biomass is heated to a temperature betweenabout 800 and 1400 degrees Fahrenheit, but no oxygen is introduced tosupport combustion resulting in the creation of gas, fuel oil andcharcoal.

Gasification: biomass can be used to produce methane through heating oranaerobic digestion. Syngas, a mixture of carbon monoxide and hydrogen,can be derived from biomass.

Landfill Gas: is generated by the decay (anaerobic digestion) of buriedgarbage in landfills. When the organic waste decomposes, it generatesgas consisting of approximately 50% methane, the major component ofnatural gas.

Anaerobic digestion: converts organic matter to a mixture of methane,the major component of natural gas, and carbon dioxide. In oneembodiment, biomass such as waterwaste (sewage), manure, or foodprocessing waste, is mixed with water and fed into a digester tankwithout air.

Fermentation

Alcohol Fermentation: fuel alcohol is produced by converting starch tosugar, fermenting the sugar to alcohol, then separating the alcoholwater mixture by distillation. Feedstocks such as wheat, barley,potatoes, and waste paper, sawdust, and straw containing sugar, starch,or cellulose can be converted to alcohol by fermentation with yeast.

Transesterification: An exemplary reaction for converting oil tobiodiesel is called transesterification. The transesterification processreacts an alcohol (like methanol) with the triglyceride oils containedin vegetable oils, animal fats, or recycled greases, forming fatty acidalkyl esters (biodiesel) and glycerin. The reaction requires heat and astrong base catalyst, such as sodium hydroxide or potassium hydroxide.

Biodiesel: Biodiesel is a mixture of fatty acid alkyl esters made fromvegetable oils, animal fats or recycled greases. Biodiesel can be usedas a fuel for vehicles in its pure form, but it is usually used as apetroleum diesel additive to reduce levels of particulates, carbonmonoxide, hydrocarbons and air toxics from diesel-powered vehicles.

Hydrolysis: includes hydrolysis of a compound, e.g., a biomass, such asa lignocellulosic material, catalyzed using an enzyme of the instantinvention.

Congeneration: is the simultaneous production of more than one form ofenergy using a single fuel and facility. In one embodiment, biomasscogeneration has more potential growth than biomass generation alonebecause cogeneration produces both heat and electricity.

In one embodiment, the polypeptides of the invention have an aldolaseactivity, including pyruvate aldolase activity, such as, withoutlimitation, HMG and/or KHG aldolase activity, or other enzymaticactivity for generating biodiesel, bioethanol, biobutanol, biopropanol,or biomethanol, from an organic material, e.g., a biomass, such ascompositions derived from plants and animals, including any agriculturalcrop or other renewable feedstock, an agricultural residue or an animalwaste, or the organic components of municipal and industrial wastes, ormicroorganisms such as algae or yeast. In one embodiment, polypeptidesof the invention are used in processes for converting lignocellulosicbiomass to ethanol, butanol, propanol, methanol or otherwise are used inprocesses for hydrolyzing or digesting biomaterials such that they canbe used as a biofuel (including bioethanol, biobutanol, biopropanol,biomethanol, or biodiesel), or for making it easier for the biomass tobe processed into a fuel. In an alternative embodiment, polypeptides ofthe invention are used in processes for a transesterification processreacting an alcohol (like methanol) with a triglyceride oil contained ina vegetable oil, animal fat or recycled greases, forming fatty acidalkyl esters (biodiesel) and glycerin. In one embodiment, biodiesel ismade from soybean oil or recycled cooking oils Animal's fats, othervegetable oils, and other recycled oils can also be used to producebiodiesel, depending on their costs and availability. In anotherembodiment, blends of all kinds of fats and oils are used to produce abiodiesel fuel of the invention.

Enzymes of the invention can also be used in glycerin refining. Theglycerin by-product contains unreacted catalyst and soaps that areneutralized with an acid. Water and alcohol are removed to produce 50%to 80% crude glycerin. The remaining contaminants include unreacted fatsand oils, which can be processes using the polypeptides of theinvention. In large biodiesel plants of the invention, the glycerin canbe further purified, e.g., to 99% or higher purity, for thepharmaceutical and cosmetic industries.

Bioethanol, biobutanol, biopropanol, biomethanol, and/or biodiesel aremade using the polypeptides of the invention can be used with fueloxygenates to improve combustion characteristics. Adding oxygen resultsin more complete combustion, which reduces carbon monoxide emissions.This is another environmental benefit of replacing petroleum fuels withbiofuels (e.g., a fuel of the invention). A bioethanol, biobutanol,biopropanol, biomethanol, and/or biodiesel made using the compositionsand/or methods of this invention can be blended with gasoline to form anE10 blend (about 5% to 10% ethanol and about 90% to 95% gasoline), butit can be used in higher concentrations such as E85 or in its pure form.A bioethanol, biobutanol, biopropanol, biomethanol, and/or biodieselmade using the compositions and/or methods of this invention can beblended with petroleum diesel to form a B20 blend (20% biodiesel and 80%petroleum diesel), although other blend levels can be used up to B100(pure biodiesel).

The invention also provides processes for making ethanol (“bioethanol”),butanol (“biobutanol”), propanol (“biopropanol”), methanol(“biomethanol”), and/or diesel (“biodiesel”) from compositionscomprising lignocellulosic biomass. The lignocellulose biomass materialcan be obtained from agricultural crops, as a byproduct of food or feedproduction, or as lignocellulosic waste products, such as plant residuesand waste paper. Examples of suitable plant sources or plant residuesfor treatment with polypeptides of the invention include kelp, algae,grains, seeds, stems, leaves, hulls, husks, corn cobs, corn stover,straw, grasses (e.g., Indian grass, such as Sorghastrum nutans; or,switch grass, e.g., Panicum species, such as Panicum virgatum), and thelike, as well as wood, wood chips, wood pulp, and sawdust. Examples ofpaper waste suitable for treatment with polypeptides of the inventioninclude discard photocopy paper, computer printer paper, notebook paper,notepad paper, typewriter paper, and the like, as well as newspapers,magazines, cardboard, and paper-based packaging materials.

In one embodiment, the enzymes and methods of the invention can be usedin conjunction with more “traditional” means of making ethanol,methanol, butanol, propanol and/or diesel from biomass, e.g., as methodscomprising hydrolyzing lignocellulosic materials by subjecting driedlignocellulosic material in a reactor to a catalyst comprised of adilute solution of a strong acid and a metal salt; this can lower theactivation energy, or the temperature, of cellulose hydrolysis to obtainhigher sugar yields; see, e.g., U.S. Pat. Nos. 6,660,506; and 6,423,145.

Another embodiment that incorporates use of enzymes of the inventioncomprises hydrolyzing lignocellulosic material containing hemicellulose,cellulose and lignin by subjecting the material to a first stagehydrolysis step in an aqueous medium at a temperature and a pressurechosen to effect primarily depolymerization of hemicellulose withoutmajor depolymerization of cellulose to glucose. This step results in aslurry in which the liquid aqueous phase contains dissolvedmonosaccharides resulting from depolymerization of hemicellulose and asolid phase containing cellulose and lignin. A second stage hydrolysisstep can comprise conditions such that at least a major portion of thecellulose is depolymerized, such step resulting in a liquid aqueousphase containing dissolved/soluble depolymerization products ofcellulose. See, e.g., U.S. Pat. No. 5,536,325. Enzymes of the inventioncan be added at any stage of this exemplary process.

Another embodiment that incorporates use of enzymes of the inventioncomprises processing a lignocellulose-containing biomass material by oneor more stages of dilute acid hydrolysis with about 0.4% to 2% strongacid; and treating an unreacted solid lignocellulosic component of theacid hydrolyzed biomass material by alkaline delignification to produceprecursors for biodegradable thermoplastics and derivatives. See, e.g.,U.S. Pat. No. 6,409,841. Enzymes of the invention can be added at anystage of this exemplary process.

Another embodiment that incorporates use of enzymes of the inventioncomprises prehydrolyzing lignocellulosic material in a prehydrolysisreactor; adding an acidic liquid to the solid lignocellulosic materialto make a mixture; heating the mixture to reaction temperature;maintaining reaction temperature for time sufficient to fractionate thelignocellulosic material into a solubilized portion containing at leastabout 20% of the lignin from the lignocellulosic material and a solidfraction containing cellulose; removing a solubilized portion from thesolid fraction while at or near reaction temperature wherein thecellulose in the solid fraction is rendered more amenable to enzymaticdigestion; and recovering a solubilized portion. See, e.g., U.S. Pat.No. 5,705,369. Enzymes of the invention can be added at any stage ofthis exemplary process.

The invention provides methods for making motor fuel compositions (e.g.,for spark ignition motors) based on liquid hydrocarbons blended with afuel grade alcohol made by using an enzyme or a method of the invention.In one embodiment, the fuels made by use of an enzyme of the inventioncomprise, e.g., coal gas liquid- or natural gas liquid-ethanol,methanol, butanol, propanol and/or diesel blends. In one embodiment, aco-solvent is biomass-derived 2-methyltetrahydrofuran (MTHF). See, e.g.,U.S. Pat. No. 6,712,866.

In one embodiment, methods of the invention for the enzymaticdegradation of lignocellulose, e.g., for production of ethanol fromlignocellulosic material, can also comprise use of ultrasonic treatmentof the biomass material; see, e.g., U.S. Pat. No. 6,333,181.

In another embodiment, methods of the invention for producingbioethanol, biobutanol, biopropanol, biomethanol, and/or biodiesel froma cellulosic substrate comprise providing a reaction mixture in the formof a slurry comprising cellulosic substrate, an enzyme of this inventionand a fermentation agent (e.g., within a reaction vessel, such as asemi-continuously solids-fed bioreactor), and the reaction mixture isreacted under conditions sufficient to initiate and maintain afermentation reaction (as described, e.g., in U.S. Pat. App. No.20060014260). In one embodiment, experiment or theoretical calculationscan determine an optimum feeding frequency. In one embodiment,additional quantities of the cellulosic substrate and the enzyme areprovided into the reaction vessel at an interval(s) according to theoptimized feeding frequency.

One exemplary process for making biofuels (such as bioethanol,biobutanol, biopropanol, biomethanol, and/or biodiesel) of the inventionis described in U.S. Pat. App. Pub. Nos. 20050069998; 20020164730; andin one embodiment comprises stages of grinding the lignocellulosicbiomass (e.g., to a size of 15-30 mm), subjecting the product obtainedto steam explosion pre-treatment (e.g., at a temperature of 190-230° C.)for between 1 and 10 minutes in a reactor; collecting the pre-treatedmaterial in a cyclone or related product of manufacture; and separatingthe liquid and solid fractions by filtration in a filter press,introducing the solid fraction in a fermentation deposit and adding oneor more enzymes of the invention, e.g., a cellulase and/orbeta-glucosidase enzyme (e.g., dissolved in citrate buffer pH 4.8).

Another exemplary process for making biofuels (such as bioethanol,biobutanol, biopropanol, biomethanol, and/or biodiesel) of the inventioncomprising using enzymes of the invention comprises pretreating astarting material comprising a lignocellulosic feedstock comprising atleast hemicellulose and cellulose. In one embodiment, the startingmaterial comprises potatoes, soybean (rapeseed), barley, rye, corn,oats, wheat, beets or sugar cane or a component or waste or food or feedproduction byproduct. The starting material (“feedstock”) is reacted atconditions which disrupt the plant's fiber structure to effect at leasta partial hydrolysis of the hemicellulose and cellulose. Disruptiveconditions can comprise, e.g., subjecting the starting material to anaverage temperature of 180° C. to 270° C. at pH 0.5 to 2.5 for a periodof about 5 seconds to 60 minutes; or, temperature of 220° C. to 270° C.,at pH 0.5 to 2.5 for a period of 5 seconds to 120 seconds, orequivalent. This generates a feedstock with increased accessibility tobeing digested by an enzyme, e.g., a cellulase enzyme of the invention.U.S. Pat. No. 6,090,595.

Exemplary conditions for hydrolysis of lignocellulosic material includereactions at temperatures between about 30° C. and 48° C., and/or a pHbetween about 4.0 and 6.0. Other exemplary conditions include atemperature between about 30° C. and 60° C. and a pH between about 4.0and 8.0.

The enzymes in accordance with the invention can catalyze reactions withexquisite stereo-, regio- and chemo-selectivities. The aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzymes in accordancewith the invention can be engineered to function in various solvents,operate at extreme pHs (for example, high pHs and low pHs) extremetemperatures (for example, high temperatures and low temperatures),extreme salinity levels (for example, high salinity and low salinity)and catalyze reactions with compounds that are structurally unrelated totheir natural, physiological substrates.

Feeds and Food or Feed and Food Additives

In addition to providing dietary aids or supplements, or foodsupplements and additives, the invention also provides compositions andmethods for treating human and animal feeds and foods and food or feedadditives using a polypeptide in accordance with the invention, such asa protein having aldolase activity, such as pyruvate aldolase, such asHMG and/or KHG aldolase enzymes in accordance with the invention, and/orthe antibodies in accordance with the invention. In some embodiments,the invention provides animal feeds, foods, and additives comprisingaldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzymes in accordance with the invention and/or antibodies in accordancewith the invention. The animal can be any farm animal or any animal.

The animal feed additive in accordance with the invention may be agranulated enzyme product that may readily be mixed with feedcomponents. Alternatively, feed additives in accordance with theinvention can form a component of a pre-mix. The granulated enzymeproduct in accordance with the invention may be coated or uncoated. Theparticle size of the enzyme granulates can be compatible with that offeed and pre-mix components. This provides a safe and convenient mean ofincorporating enzymes into feeds. Alternatively, the animal feedadditive in accordance with the invention may be a stabilized liquidcomposition. This may be an aqueous or oil-based slurry. See U.S. Pat.No. 6,245,546.

Aldolase, such as pyruvate aldolase, such as HMG and/or KHG aldolaseenzymes of the present invention, in the modification of feed or a food,can process the food or feed either in vitro (by modifying components ofthe feed or food) or in vivo. Polypeptides in accordance with theinvention can be added to feed or food compositions.

In some embodiments, an enzyme in accordance with the invention is addedin combination with another enzyme, such as beta-galactosidases,catalases, laccases, cellulases, other aldolases, endoglycosidases,endo-beta-1,4-laccases, amyloglucosidases, glucosidases, glucoseisomerases, glycosyltransferases, lipases, phospholipases,lipooxygenases, beta-laccases, endo-beta-1,3(4)-laccases, cutinases,peroxidases, amylases, phytases, glucoamylases, pectinases, reductases,oxidases, decarboxylases, phenoloxidases, ligninases, pullulanases,arabinanases, hemicellulases, mannanases, xylolaccases, xylanases,pectin acetyl esterases, rhamnogalacturonan acetyl esterases, proteases,peptidases, proteinases, polygalacturonases, rhamnogalacturonases,galactanases, pectin lyases, transglutaminases, pectin methylesterases,cellobiohydrolases and/or transglutaminases. These enzyme digestionproducts are more digestible by the animal. Thus, aldolase, such aspyruvate aldolase, such as HMG and/or KHG aldolase enzymes in accordancewith the invention can contribute to the available energy of the feed orfood, or to the digestibility of the food or feed by breaking downcellulose.

In other embodiments, aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzyme in accordance with the invention can besupplied by expressing the enzymes directly in transgenic feed crops(as, such as transgenic plants, seeds and the like), such as grains,cereals, corn, soy bean, rape seed, lupin and the like. As discussedabove, the invention provides transgenic plants, plant parts and plantcells comprising a nucleic acid sequence encoding a polypeptide inaccordance with the invention. In some embodiments, the nucleic acid isexpressed such that the aldolase, such as pyruvate aldolase, such as HMGand/or KHG aldolase enzyme in accordance with the invention is producedin recoverable quantities. The aldolase, such as pyruvate aldolase, suchas HMG and/or KHG aldolase enzyme can be recovered from any plant orplant part. Alternatively, the plant or plant part containing therecombinant polypeptide can be used as such for improving the quality ofa food or feed, such as improving nutritional value, palatability, etc.

In some embodiments, the enzyme delivery matrix in accordance with theinvention is in the form of discrete plural particles, pellets orgranules. By “granules” is meant particles that are compressed orcompacted, such as by a pelletizing, extrusion, or similar compacting toremove water from the matrix. Such compression or compacting of theparticles also promotes intraparticle cohesion of the particles. Forexample, the granules can be prepared by pelletizing the grain-basedsubstrate in a pellet mill. The pellets prepared thereby are ground orcrumbled to a granule size suitable for use as an adjuvant in animalfeed. Because the matrix is itself approved for use in animal feed, itcan be used as a diluent for delivery of enzymes in animal feed.

In some embodiments, the aldolase, such as pyruvate aldolase, such asHMG and/or KHG aldolase enzyme contained in the invention enzymedelivery matrix and methods is a thermostable aldolase, such as pyruvatealdolase, such as HMG and/or KHG aldolase enzyme, as described herein,so as to resist inactivation of the aldolase, such as pyruvate aldolase,such as HMG and/or KHG aldolase enzyme during manufacture where elevatedtemperatures and/or steam may be employed to prepare the palletizedenzyme delivery matrix. During digestion of feed containing theinvention enzyme delivery matrix, aqueous digestive fluids will causerelease of the active enzyme. Other types of thermostable enzymes andnutritional supplements that are thermostable can also be incorporatedin the delivery matrix for release under any type of aqueous conditions.

In some embodiments, a coating is applied to the enzyme matrix particlesfor many different purposes, such as to add a flavor or nutritionsupplement to animal feed, to delay release of animal feed supplementsand enzymes in gastric conditions, and the like. In some embodiments,the coating is applied to achieve a functional goal, for example,whenever it is desirable to slow release of the enzyme from the matrixparticles or to control the conditions under which the enzyme will bereleased. The composition of the coating material can be such that it isselectively broken down by an agent to which it is susceptible (such asheat, acid or base, enzymes or other chemicals). Alternatively, two ormore coatings susceptible to different such breakdown agents may beconsecutively applied to the matrix particles.

The invention is also directed towards a process for preparing anenzyme-releasing matrix. In accordance with the invention, the processcomprises providing discrete plural particles of a grain-based substratein a particle size suitable for use as an enzyme-releasing matrix,wherein the particles comprise an aldolase, such as pyruvate aldolase,HMG and/or KHG aldolase enzyme encoded by an amino acid sequence inaccordance with the invention. In some embodiments, the process includescompacting or compressing the particles of enzyme-releasing matrix intogranules, which most In some embodiments is accomplished by pelletizing.The mold inhibitor and cohesiveness agent, when used, can be added atany suitable time, and, in some embodiments are mixed with thegrain-based substrate in the desired proportions prior to pelletizing ofthe grain-based substrate. Moisture content in the pellet mill feed insome embodiments is in the ranges set forth above with respect to themoisture content in the finished product, and, in some embodiments, isabout 14-15%. In some embodiments, moisture is added to the feedstock inthe form of an aqueous preparation of the enzyme to bring the feedstockto this moisture content. The temperature in the pellet mill in someembodiments is brought to about 82° C. with steam. The pellet mill maybe operated under any conditions that impart sufficient work to thefeedstock to provide pellets. The pelleting process itself is acost-effective process for removing water from the enzyme-containingcomposition.

The compositions and methods in accordance with the invention can bepracticed in conjunction with administration of prebiotics, which arehigh molecular weight sugars, such as fructo-oligosaccharides (FOS);galacto-oligosaccharides (GOS), GRAS (Generally Recognized As Safe)material. These prebiotics can be metabolized by some probiotic lacticacid bacteria (LAB). They are non-digestible by the majority ofintestinal microbes.

Treating Foods and Food Processing

The invention provides foods and feeds comprising enzymes in accordancewith the invention, and methods for using enzymes in accordance with theinvention in processing foods and feeds. Aldolase, such as pyruvatealdolase, HMG and/or KHG aldolase enzymes in accordance with theinvention have numerous applications in food processing industry. Insome embodiments, the invention provides methods for hydrolyzingcellulose-comprising compositions, including, such as a plant cell, abacterial cell, a yeast cell, an insect cell, or an animal cell, or anyplant or plant part, or any food or feed, a waste product and the like.

For example, the invention provides feeds or foods comprising analdolase, such as pyruvate aldolase, HMG and/or KHG aldolase enzyme theinvention, such as in a feed, a liquid, such as a beverage (such as afruit juice or a beer), a bread or a dough or a bread product, or adrink (such as a beer) or a beverage precursor (such as a wort).

The food treatment processes in accordance with the invention can alsoinclude the use of any combination of other enzymes such astryptophanases or tyrosine decarboxylases, laccases, catalases,laccases, other aldolases, cellulases, endoglycosidases,endo-beta-1,4-laccases, amyloglucosidases, glucosidases, glucoseisomerases, glycosyltransferases, lipases, phospholipases,lipooxygenases, beta-laccases, endo-beta-1,3(4)-laccases, cutinases,peroxidases, amylases, phytases, glucoamylases, pectinases, reductases,oxidases, decarboxylases, phenoloxidases, ligninases, pullulanases,arabinanases, hemicellulases, mannanases, xylolaccases, xylanases,pectin acetyl esterases, rhamnogalacturonan acetyl esterases, proteases,peptidases, proteinases, polygalacturonases, rhamnogalacturonases,galactanases, pectin lyases, transglutaminases, pectin methylesterases,cellobiohydrolases and/or transglutaminases.

Pharmaceutical Compositions and Dietary Supplements

The invention also provides pharmaceutical compositions and dietarysupplements (such as dietary aids) comprising an aldolase in accordancewith the invention. The aldolase activity comprises pyruvate aldolase,HMG and/or KHG aldolase activity. In some embodiments, thepharmaceutical compositions and dietary supplements (such as dietaryaids) are formulated for oral ingestion.

Periodontal treatment compounds can comprise an enzyme in accordancewith the invention, such as described in U.S. Pat. No. 6,776,979.Compositions and methods for the treatment or prophylaxis of acidic gutsyndrome can comprise an enzyme in accordance with the invention, suchas described in U.S. Pat. No. 6,468,964.

In other embodiments, wound dressings, implants and the like compriseantimicrobial (such as antibiotic-acting) enzymes, including an enzymein accordance with the invention (including, such as sequences inaccordance with the invention). Enzymes in accordance with the inventioncan also be used in alginate dressings, antimicrobial barrier dressings,burn dressings, compression bandages, diagnostic tools, gel dressings,hydro-selective dressings, hydrocellular (foam) dressings, hydrocolloiddressings, I.V dressings, incise drapes, low adherent dressings, odorabsorbing dressings, paste bandages, post operative dressings, scarmanagement, skin care, transparent film dressings and/or wound closure.Enzymes in accordance with the invention can be used in wound cleansing,wound bed preparation, to treat pressure ulcers, leg ulcers, burns,diabetic foot ulcers, scars, IV fixation, surgical wounds and minorwounds. Enzymes in accordance with the invention can be used to insterile enzymatic debriding compositions, such as ointments. In variousembodiments, the aldolase is formulated as a tablet, gel, pill, implant,liquid, spray, film, micelle, powder, food, feed pellet or as anencapsulated formulation.

The pharmaceutical compositions and dietary supplements in accordancewith the invention can also include the use of any combination of otherenzymes such as beta-galactosidases, catalases, laccases, cellulases,other aldolases, endoglycosidases, endo-beta-1,4-laccases,amyloglucosidases, glucosidases, glucose isomerases,glycosyltransferases, lipases, phospholipases, lipooxygenases,beta-laccases, endo-beta-1,3(4)-laccases, cutinases, peroxidases,amylases, phytases, glucoamylases, pectinases, reductases, oxidases,decarboxylases, phenoloxidases, ligninases, pullulanases, arabinanases,hemicellulases, mannanases, xylolaccases, xylanases, pectin acetylesterases, rhamnogalacturonan acetyl esterases, proteases, peptidases,proteinases, polygalacturonases, rhamnogalacturonases, galactanases,pectin lyases, transglutaminases, pectin methylesterases,cellobiohydrolases and/or transglutaminases.

Biosynthetic Pathways to Produce R,R and Other Stereoisomers of Monatin

As described, inter alia, in WO 03/091396 A2 (see FIGS. 1-3 and 11-13),monatin can be produced from tryptophan through a multi-step pathwayinvolving biological conversions (i.e. facilitating the reaction of asubstrate to a product with a polypeptide). A pathway described involvesbiologically converting tryptophan to indole-3-pyruvate, biologicallyconverting indole-3-pyruvate to 2-hydroxy 2-(indol-3-ylmethyl)-4-ketoglutaric acid (“MP”), and biologically converting MP to monatin. In someembodiments, polypeptides of the invention can be used to facilitate thereaction of indole-3-pyruvate to form MP. In some embodiments,polypeptides of the invention can be used to preferentially facilitatethe production of R-MP.

In some embodiments, one or more polypeptides chosen from isolated orrecombinant polypeptides of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ IDNO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ IDNO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ IDNO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116,SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ IDNO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144,SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ IDNO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172,SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ IDNO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200,SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ IDNO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228,SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ IDNO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256,SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ IDNO:266, SEQ ID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:284,SEQ ID NO:286, SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:292, SEQ IDNO:294, SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ ID NO:302, SEQID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312,SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ IDNO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQID NO:332, or SEQ ID NO:334, or fragments or subsequences thereof havingaldolase activity may be useful in facilitating a reaction within amulti-step pathway to produce a product chosen from monatin, monatinderivatives, salts thereof and combinations thereof. In one embodiment,the polypeptides with aldolase activity may be useful in facilitating areaction in which indole-3-pyruvate is converted to MP as one stepwithin a multi-step pathway to produce a product chosen from monatin,monatin derivatives, salts thereof and combinations thereof.

In another embodiment, one or more polypeptides chosen from isolated orrecombinant polypeptides with HMG aldolase activity of any of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52,SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62,SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72,SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82,SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92,SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102,SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ IDNO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130,SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ IDNO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158,SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ IDNO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186,SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ IDNO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214,SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ IDNO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242,SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ IDNO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258, SEQ ID NO:260, SEQID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQ ID NO:268, SEQ ID NO:270,SEQ ID NO:272, SEQ ID NO:274, SEQ ID NO:276, SEQ ID NO:278, SEQ IDNO:280, SEQ ID NO:282, SEQ ID NO:284, SEQ ID NO:286, SEQ ID NO:288, SEQID NO:290, SEQ ID NO:292, SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298,SEQ ID NO:300, SEQ ID NO:302, SEQ ID NO:304 or fragments or subsequencesthereof having aldolase activity may be useful in facilitating areaction between indole-3-pyruvate and a C3 carbon source as one stepwithin a multi-step pathway to produce a product chosen from monatin,monatin derivatives, salts thereof and combinations thereof. In oneembodiment, the polypeptides with HMG aldolase activity may be useful infacilitating a reaction in which indole-3-pyruvate is converted to MP asone step within a multi-step pathway to produce a product chosen frommonatin, monatin derivatives, salts thereof and combinations thereof.

In yet another embodiment, one or more polypeptides chosen from isolatedor recombinant polypeptides with KHG aldolase activity of any of SEQ IDNO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324,SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, or SEQ IDNO:334 or fragments or subsequences thereof having aldolase activity maybe useful in facilitating a reaction between indole-3-pyruvate and a C3carbon source as one step within a multi-step pathway to produce aproduct chosen from monatin, monatin derivatives, salts thereof andcombinations thereof. In one embodiment, the polypeptides with KHGaldolase activity may be useful in facilitating a reaction in whichindole-3-pyruvate is converted to MP as one step within a multi-steppathway to produce a product chosen from monatin, monatin derivatives,salts thereof and combinations thereof.

Additionally, one or more polypeptides encoded by one or more nucleicacids sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more, or complete (100%) sequence identity to a nucleic acid inaccordance with the invention, including SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ IDNO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151,SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ IDNO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179,SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ IDNO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207,SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ IDNO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235,SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ IDNO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263,SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ IDNO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291,SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ IDNO:301, SEQ ID NO:303, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319,SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ IDNO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ ID NO:335, SEQ ID NO:336, SEQID NO:337, and SEQ ID NO:338 over a region of at least about 10, 15, 20,25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750,1800, 1850, 1900, 1950, 2000, 2050, 2100, 2200, 2250, 2300, 2350, 2400,2450, 2500, or more residues may be useful in facilitating a reactionbetween indole-3-pyruvate and a C3 carbon source as one step within amulti-step pathway to produce a product chosen from monatin, monatinderivatives, salts thereof and combinations thereof. In one embodiment,the one or more polypeptides, or fragments or subsequences thereof withaldolase activity may be useful in facilitating a reaction in whichindole-3-pyruvate is converted to MP as one step within a multi-steppathway to produce a product chosen from monatin, monatin derivatives,salts thereof and combinations thereof.

In another embodiment of the invention, one or more polypeptides withHMG aldolase activity encoded by a nucleic acid sequence having at leastabout 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)sequence identity to a nucleic acid in accordance with the invention,including SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117,SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ IDNO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145,SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ IDNO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173,SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ IDNO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201,SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ IDNO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229,SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ IDNO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257,SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ IDNO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:285,SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ IDNO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQID NO:305 over a region of at least about 10, 15, 20, 25, 30, 35, 40,45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900,1950, 2000, 2050, 2100, 2200, 2250, 2300, 2350, 2400, 2450, 2500, ormore residues may be useful in facilitating a reaction betweenindole-3-pyruvate and a C3 carbon source as one step within a multi-steppathway to produce a product chosen from monatin, monatin derivatives,salts thereof and combinations thereof. In one embodiment, the one ormore polypeptides with HMG aldolase activity may be useful infacilitating a reaction in which indole-3-pyruvate is converted to MP asone step within a multi-step pathway to produce a product chosen frommonatin, monatin derivatives, salts thereof and combinations thereof.

In yet another embodiment of the invention, one or more polypeptideswith KHG aldolase activity encoded by a nucleic acid sequence having atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete(100%) sequence identity to a nucleic acid in accordance with theinvention, including SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ IDNO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321, SEQID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ ID NO:331,SEQ ID NO:333, SEQ ID NO:335, SEQ ID NO:336, SEQ ID NO:337, and SEQ IDNO:338 over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45,50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950,2000, 2050, 2100, 2200, 2250, 2300, 2350, 2400, 2450, 2500, or moreresidues may be useful in facilitating a reaction betweenindole-3-pyruvate and a C3 carbon source as one step within a multi-steppathway to produce a product chosen from monatin, monatin derivatives,salts thereof and combinations thereof. In one embodiment, the one ormore polypeptides with KHG aldolase activity may be useful infacilitating a reaction in which indole-3-pyruvate is converted to MP asone step within a multi-step pathway to produce a product chosen frommonatin, monatin derivatives, salts thereof and combinations thereof.

Furthermore, one or more polypeptides with aldolase activity encoded bya nucleic acid sequence that hybridizes under stringent condition to anucleic acid of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117,SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ IDNO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145,SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ IDNO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173,SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ IDNO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201,SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ IDNO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229,SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ IDNO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257,SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ IDNO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:285,SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ IDNO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ ID NO:313,SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321, SEQ IDNO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQID NO:333, SEQ ID NO:335, SEQ ID NO:336, SEQ ID NO:337, and SEQ IDNO:338 may be useful in facilitating a reaction betweenindole-3-pyruvate and a C3 carbon source as one step within a multi-steppathway to produce a product chosen from monatin, monatin derivatives,salts thereof and combinations thereof. In one embodiment, the one ormore polypeptides with aldolase activity may be useful in facilitating areaction in which indole-3-pyruvate is converted to MP as one stepwithin a multi-step pathway to produce a product chosen from monatin,monatin derivatives, salts thereof and combinations thereof.

In another embodiment of the invention, one or more polypeptides withHMG aldolase activity encoded by a nucleic acid sequence that hybridizesunder stringent condition to a nucleic acid of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ IDNO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151,SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ IDNO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179,SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ IDNO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207,SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ IDNO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235,SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ IDNO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263,SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ IDNO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291,SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ IDNO:301, SEQ ID NO:303, SEQ ID NO:305 may be useful in facilitating areaction between indole-3-pyruvate and a C3 carbon source as one stepwithin a multi-step pathway to produce a product chosen from monatin,monatin derivatives, salts thereof and combinations thereof. In oneembodiment, the one or more polypeptides with HMG aldolase activity maybe useful in facilitating a reaction in which indole-3-pyruvate isconverted to MP as one step within a multi-step pathway to produce aproduct chosen from monatin, monatin derivatives, salts thereof andcombinations thereof.

In yet another embodiment of the invention, one or more polypeptideswith KHG aldolase activity encoded by a nucleic acid sequence thathybridizes under stringent condition to a nucleic acid of SEQ ID NO:307,SEQ ID NO:309, SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQ IDNO:317, SEQ ID NO:319, SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325, SEQID NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ ID NO:335,SEQ ID NO:336, SEQ ID NO:337, and SEQ ID NO:338 may be useful infacilitating a reaction between indole-3-pyruvate and a C3 carbon sourceas one step within a multi-step pathway to produce a product chosen frommonatin, monatin derivatives, salts thereof and combinations thereof. Inone embodiment, the one or more polypeptides with KHG aldolase activitymay be useful in facilitating a reaction in which indole-3-pyruvate isconverted to MP as one step within a multi-step pathway to produce aproduct chosen from monatin, monatin derivatives, salts thereof andcombinations thereof.

The polypeptides with aldolase activity described herein may be usefulin facilitating a reaction between indole-3-pyruvate and a C3 carbonsource. The C3 carbon source may be, but is not limited to,oxaloacetate, pyruvate or a pyruvate derivative, such asphosphoenolpyruvate. In one embodiment, the C3 carbon source ispyruvate.

Exemplary enzymes useful for the conversion of the reaction productbetween indole-3-pyruvate and the C3 carbon source to monatin includemembers of the enzyme classes: tryptophan aminotransferases (2.6.1.27),tryptophan dehydrogenases (1.4.1.19), D-amino acid dehydrogenases(1.4.99.1), glutamate dehydrogenases (1.4.1.2-4), phenylalaninedehydrogenase (EC 1.4.1.20), tryptophan-phenylpyruvate transaminases(2.6.1.28), or more generally members of the aminotransferase family(2.6.1.-) such as aspartate aminotransferase (EC 2.6.1.1), tyrosine(aromatic) aminotransferase (2.6.1.5), D-tryptophan aminotransferase, orD-alanine (2.6.1.21) aminotransferase (see FIG. 2 of WO 03/091396 A2).This reaction can also be performed using chemical reactions. Aminationof the keto acid (MP) is performed by reductive amination using ammoniaand sodium cyanoborohydride. FIGS. 11-13 of WO 03/091396 A2 showadditional polypeptides that can be used to convert MP to monatin, aswell as providing increased yields of monatin from indole-3-pyruvate ortryptophan. In one embodiment, these enzymes are utilized to catalyzethe conversion of MP, the reaction product between indole-3-pyruvate andpyruvate, to monatin.

The taste profile of a monatin composition can be altered by controllingthe relative amount of the various stereoisomers of monatin in thecomposition. The present disclosure provides pathways and substances forproducing monatin compositions with a desired percentage of R,R monatinand/or S,R monatin.

The chirality of the monatin compounds produced by the pathwaysdisclosed can be affected both by pH and by the polypeptides used forthe biological conversions. The polypeptides with aldolase activitydescribed herein, may be utilized to control the chirality of themonatin carbon-2 (see Formula I, above) in the reaction in whichindole-3-pyruvate is converted to MP.

Once the reaction product of the reaction between indole-3-pyruvate andthe C3 carbon source is produced, the amino group can be addedstereospecifically. Either the R or S configuration of carbon-4 (seeFormula I above) can be generated depending on whether a D- orL-aromatic acid aminotransferase is used. Many aminotransferases arespecific for the L-isomer, however, D-tryptophan aminotransferases existin certain plants (Kohiba and Mito, Proceedings of the 8th InternationalSymposium on Vitamin B₆ and Carbonyl Catalysis, Osaka, Japan 1990).Moreover, D-alanine aminotransferases (2.6.1.21), D-methionine-pyruvateaminotransferases (2.6.1.41) and both (R)-3-amino-2-methylpropanoateaminotransferase (2.6.1.61), (S)-3-amino-2-methylpropanoateaminotransferase (2.6.1.22), and D-phenylglycine aminotransferase havebeen identified. Certain aminotransferases may only accept the substratefor this reaction with a particular configuration at the C2 carbon.Therefore, even if the conversion to the reaction product betweenindole-3-pyruvate and the C3 carbon source is not stereospecific, thestereochemistry of the final product can be controlled through theappropriate selection of an aminotransferase. Because the reactions arereversible, the unreacted reaction product (undesired isomer) can berecycled back to its constituents and a racemic mixture of the reactionproduct can be reformed.

An example of a suitable amino donor for the addition of an amino groupto the reaction product of the reaction between the indole-3-pyruvateand the C3 carbon source includes, but is not limited to an amino acid,such as alanine, aspartate, lysine, glutamate, glycine, and tryptophan.

Referring now to the figures, the following should be noted. The flowcharts identify pathways for producing monatin, but are not limited toany particular method for practicing the pathways. For example, thepathways may be practiced in vivo, in vitro, or a combination thereof.

Furthermore, practice of the pathways does not require that each of theidentified components (such as reactants and enzymes) is explicitlyprovided by the practitioner, so long as sufficient components, orsources of components, and reaction conditions are provided so that thepathway can potentially proceed. In other words, for example, if afigure depicts a process for producing a monatin composition, whichincludes producing indole-3-pyruvate from L-tryptophan, producing2-hydroxy 2-(indol-3ylmethyl)-4-keto glutaric acid (“monatin precursor”or “MP”) from indole-3-pyruvate, and producing monatin from MP, whereineach reaction is facilitated by an appropriate enzyme, it iscontemplated that practice of that pathway includes combiningL-tryptophan with α-ketoglutarate and enzymes contemplated forfacilitating the identified reactions, and under conditions suitable foreach of the reactions to occur without also explicitly providingindole-3-pyruvate or MP. In such an instance L-tryptophan could reactwith α-ketoglutarate to produce indole-3-pyruvate. Due to the setconditions and the provided enzyme, the indole-3-pyruvate produced fromthe L-tryptophan reaction could react to form MP, and then due to theset conditions and the provided enzyme, the MP produced from theindole-3-pyruvate reaction could react to form monatin.

It should also be noted that practice of the depicted pathways does notrequire the practitioner to explicitly provide the identified startingmaterials or enzymes. In other words, it is contemplated that practiceof any pathways which identifies L-tryptophan as a starting materialwould include providing a compound that can produce L-tryptophan, underconditions suitable for L-tryptophan production to occur and combiningthat compound with enzymes capable of facilitating the series ofreactions set forth under conditions which would be suitable for thosereactions to occur. As another example, it is also contemplated thatpracticing the identified pathway would include providing amicroorganism genetically engineered to produce monatin according to thedescribed pathway, and providing appropriate conditions for thefermentation process to occur. For example, a microorganism, whichnaturally produces large amounts of L-tryptophan could be geneticallyengineered to produce or over-produce one or more of the enzymes used tofacilitate reactions in the pathway to monatin, and appropriateconditions could be provided so that the microorganism would therebyproduce monatin.

FIG. 1 identifies the particular embodiment wherein an R-specificaldolase facilitates the reaction of indole-3-pyruvate and pyruvate toform R-MP. The flow chart of FIG. 1 schematically depicts a process inaccordance with the invention for making a monatin composition includingR,R monatin. As shown in FIG. 1, the overall pathway involves a reactionof tryptophan to form indole-3-pyruvate, a reaction of indole-3-pyruvateto produce MP, and a reaction of MP to produce monatin, including R,Rmonatin.

FIG. 1 further illustrates specific permutations of this overallpathway, designed to increase the production of the R,R form of monatinat the expense of the S,S, R,S and S,R forms of monatin. In particular,FIG. 1 illustrates the embodiment wherein: the aminotransferase enzymeutilized in the L-tryptophan reaction has greater activity and/orspecificity for that reaction versus the reactions of MP and 4S monatinor the oxidase has greater activity and/or specificity for L-tryptophanthan for 4R monatin; the enzyme which facilitates the reaction ofindole-3-pyruvate is a polypeptide with aldolase activity disclosedherein, and, the enzyme which facilitates the reaction of MP is a broadspecificity D-enzyme, preferably evolved to work more efficiently withthe R isomer of MP.

FIG. 1 also illustrates particular permutations designed to make theproduction of R,R monatin more economical. For example, in FIG. 1,L-tryptophan—as opposed to D-tryptophan or combinations of L- andD-tryptophan—is identified as the starting material. While the choice ofthe specific form of tryptophan does not impact the chirality of theultimate monatin compounds in the monatin composition (because thetryptophan reaction forms indole-3-pyruvate, which has no chirality),some may prefer utilizing L-tryptophan as a starting material at leastbecause L-tryptophan is currently less expensive and more easilyobtainable than D-tryptophan

Focusing now on the first reaction shown in FIG. 1, when tryptophan isconverted to indole-3-pyruvate any one or more of alpha-ketoglutarate,oxaloacetate, and pyruvate reacts to form an amino acid (glutamate,aspartate, and alanine respectively). FIG. 1 depicts the embodimentwherein the tryptophan starting material is L-tryptophan, and thealpha-ketoglutarate, oxaloacetate, and/or pyruvate produce the L-isomerform of the amino acid (such as L-glutamate, L-aspartate, and/orL-alanine, respectively).

As shown in FIG. 1, an approach to enhancing the production of R,Rmonatin involves facilitating the reaction of L-tryptophan with anenzyme having greater specificity, greater activity, or both fortryptophan as opposed to MP or monatin, and facilitating the reaction ofMP with a D-enzyme. As is disclosed in WO 03/091396 A2, certain enzymescan facilitate the reaction of tryptophan to produce indole-3-pyruvate,as well as the amination reaction of MP to produce monatin. Use of anL-aminotransferase in the amination step creates an S chiral center atthe monatin C-4 position, whereas use of a D-enzyme creates a D chiralcenter at the monatin C-4 position. Thus, in the instance where anL-aminotransferase, which facilitates the tryptophan reaction, is alsoactive in the MP reaction, R,S and S,S monatin can be formed, dependingon the form of MP present. In addition, certain other enzymes—theL-amino acid oxidases—can not only facilitate the reaction of tryptophanto indole-3-pyruvate, but may have a side activity for the degradationof R,R monatin. According to some embodiments, this 4R side activity isminimized or eliminated. An oxidase side activity on 4S forms of monatinwould decrease or minimize them from the final product and could bedesirable depending on the final composition desired. Consequently, thegreater the specificity and/or activity of the L-enzyme chosen fortryptophan versus the MP or monatin, the greater the amount of R,R andS,R produced versus S,S and R,S monatin.

Suitable enzymes for the tryptophan reaction, in accordance with theembodiment illustrated in FIG. 1, include: L-aminotransferases capableof facilitating a reaction of L-tryptophan to form indole-3-pyruvate,and which have greater specificity for that reaction over the reactionof R-MP to form 4S isomers of monatin; and, L-amino acid oxidasescapable of facilitating a reaction of L-tryptophan to formindole-3-pyruvate, and which have greater specificity and/or activityfor that reaction versus the reaction of 4R isomers of monatin to formMP, and functional equivalents of any of the foregoing. Morespecifically, non-limiting examples of suitable enzymes can be chosenfrom L-tryptophan aminotransferases (E.C. 2.6.1.27) and tyrosine(aromatic) aminotransferases (EC 2.6.1.5) and L-amino acid oxidases (EC1.4.3.2), and mutants derived from enzymes having aspartateaminotransferase activity.

Example 16 identifies a specific enzyme, a mutant HEXaspC polypeptidewhich includes a Pro 9 to Tyr substitution and an Arg 122 to Glysubstitution useful for facilitating the reactions of L-tryptophan andα-KG, oxaloacetate, pyruvate, or combinations thereof to formindole-3-pyruvate and L-glutamate, L-aspartate, and L-alanine,respectively. Another specific enzyme having “limited” activity is TatA,the L-tryptophan aminotransferase from S. meliloti. Other enzymessuitable for the tryptophan reaction in accordance with preferredembodiments of the pathway shown in FIG. 1 include those with thefollowing characteristics: an enzyme that transaminates MP at 1/10 therate or less than the rate of L-tryptophan as in Example 16 or an enzymewhen used with a racemase, as in Example 18, that produces greater than90% of the 4R isomers of monatin.

Examples of enzymes not having greater specificity for the L-tryptophanto indole-3-pyruvate conversion compared to the MP to monatin conversioninclude: HEXAspC (Example 16), Leishmania major broad specificityaminotransferase (WO 03/091396 A2), the Porcine aminotransferase (WO03/091396 A2) and Rhodobacter sphaeroides TatA (Example 18). Theseenzymes may, however, be evolved, for example through mutagenesis tohave limited activity for R-MP and/or R,R monatin versus tryptophan.

Focusing now on the second reaction identified in FIG. 1, the choice ofenzyme for facilitating the reaction of indole-3-pyruvate to MPinfluences the relative amount of R,R monatin versus S,R monatinproduced. In general, the greater the relative amount of R-MP versusS-MP produced, the greater the relative amount of R,R monatin versus S,Rmonatin produced (when a D-enzyme facilitates the reaction of MP tomonatin). Where a monatin composition having the R,R form of monatin asits only monatin component is desired, an enzyme that selectivelyproduces R-MP as opposed to S-MP (an “R-specific enzyme”) should beused. The polypeptides with aldolase activity described herein areuseful in selectively producing R-MP, as opposed to S-MP. Severalexamples of highly R-specific aldolase enzymes are demonstrated in Table1, above, Examples 4, 5 and 6, below, and in the Sequence Listing.

Focusing now on the last step of the pathway identified in FIG. 1, thereaction of R-MP to form R,R monatin is shown to be facilitated by abroad specificity D-aminotransferase, for example D-alanineaminotransferase (E.C. 2.6.1.21, also known as D-amino acidaminotransferase or D-aspartate aminotransferase) or a D-amino aciddehydrogenase. As discussed above, the conversion of MP to monatin is anamination reaction, which creates a chiral center at the monatin C-4carbon. Where the R-chiral form is desired at the C-4 position, enzymesshould be used which produce “R” chiral centers in amino acids.

According to some embodiments, the D-aminotransferase has greaterspecificity, greater activity, or both for the R-MP than forindole-3-pyruvate. According to some embodiments, the D-aminotransferasehas limited activity for the indole-3-pyruvate. Enzymes with suchcharacteristics may be evolved or mutated from existing enzymes, forexample as shown in Example 16.

Examples 9 to 12 illustrate the production of R,R-monatin fromD-tryptophan.

FIG. 2 illustrates a method of producing R,R monatin and S,R monatin.Whereas in the embodiment of FIG. 1, the aldolase used in the reactionof indole-3-pyruvate to form R-MP influences the ratio of R,R:S,Rformed, in the embodiment of FIG. 2, the D-enzyme that facilitates theconversion of MP to monatin influences the ratio of R,R:S,R formed.According to the pathway of FIG. 2, if a non-stereospecific enzyme isused to facilitate the conversion of indole-3-pyruvate to MP, then bothS-MP and R-MP can be formed. If a non-stereoselective aldolase isutilized to convert indole-3-pyruvate to MP, then a stereoselectivetransaminase is required to convert the MP to either R,R monatin or S,Rmonatin. As shown on FIG. 2, use of a D-aminotransferase or D-amino aciddehydrogenase that is stereospecific for R-MP results in the productionof R,R monatin.

FIG. 3 illustrates another alternative pathway for targeting productionof R,R monatin. The pathway of FIG. 3 is a modification of the pathwayof FIG. 1, wherein indole-3-pyruvate is produced indirectly, rather thandirectly, from L-tryptophan. More specifically, L-tryptophan isconverted to D-tryptophan, and D-tryptophan is then converted toindole-3-pyruvate.

The conversion of L-tryptophan to D-tryptophan can be facilitated by atryptophan racemase or functional equivalent thereof. Example 15provides potential sources of tryptophan racemases and screening methodsfor identifying such enzymes. It is also contemplated a tryptophanracemase may be evolved (such as via mutagenesis or recombinantengineering) for improved performance from an existing amino acidracemase.

Non-limiting examples of tryptophan racemases include homolog or mutantsof amino acid racemases (EC 5.1.1.-), for example serine racemase,wherein the homologs or mutants are capable of converting L-tryptophanto D-tryptophan. Non-limiting examples of sources from which the aminoacid racemase may be derived include: microorganisms such as Salmonellatyphimurium, Escherichia coli, Bacillus subtilis, Pseudomonasaeruginosa, Vibrio cholerae, Schizosaccaroyces pombe, Bacillus cereus,Enterococcus gallinarum, Pediococcus pentosaceus, Bacillus pumilus,Lactobacillus fermenti, Lactobacillus brevis, Aquifex pyrophilus,Lactobacilli, Streptococcus, Anabaena sp., Pseudomonas striata, Lentinusedodes, Scapharca brouhtonii Desulfurococcus sp., Thermococcus sp., andPseudomonas striata. Additional non-limiting examples of sources fromwhich the amino acid racemase may be derived include silkworm, ratbrain, or mouse brain.

Non-limiting examples of potential sources from which suitabletryptophan racemases may be derived include: microorganisms such asPseudomonas, for example Pseudomonas chlororaphis (Pseudomonasaurereofaciens) (ATCC15926), and Burkholderia pyrrocina (ATCC15958).Additional non-limiting examples of potential sources from whichsuitable tryptophan racemases may be derived include plants, for exampletobacco plants, such as Nicotiana tabacum, wheat plants, such asTriticum aestivum, beets, tomatoes, and Sclerochiton ilicifolius.

The pathway shown in FIG. 3 has certain benefits, including that evenwhere R,R monatin is the desired product, the same enzyme may be usedfor the reaction producing indole-3-pyruvate as for the reactionproducing monatin. That is, in the pathway illustrated in FIG. 1, anL-aminotransferase (or suitable L-enzyme) facilitates the reactionproducing indole-3-pyruvate, but a D-aminotransferase facilitates thereaction producing monatin. By contrast in the pathway of FIG. 3,certain D-aminotransferase that facilitates the reaction producingindole-3-pyruvate, can also facilitate the reaction producing monatin.Consequently, in pathways according to FIG. 3 broad specificityD-aminotransferases may be preferred where there is a desire to use thesame enzyme for the reaction forming indole-3-pyruvate as for thereaction forming monatin. By contrast, in pathways according to FIGS. 1,2, 4, 6, 7, and 8 production of monatin may proceed forward moreefficiently when a D-aminotransferase is chosen that has limitedactivity and/or specificity for indole-3-pyruvate as compared to R-MP.

Another benefit of the pathway schematically represented in FIG. 3 isthat the amino acid product of the reaction coupled to the reactionproducing indole-3-pyruvate can now be used as a starting material inthe reaction coupled to the reaction producing monatin. That is, in thepathway illustrated in FIG. 1, L-tryptophan reacts to produceindole-3-pyruvate and at the same time oxaloacetate, alpha-ketoglutarateand/or pyruvate react to produce an L-amino acid. Because the reactionof R-MP to form monatin is coupled with a reaction utilizing a D-aminoacid as a substrate, the L-amino acid of the reaction formingindole-3-pyruvate is not, under the conditions shown, recycled for usein the reaction coupled to the R-MP reaction. By contrast, in thepathway illustrated in FIG. 3, the reaction of D-tryptophan to formindole-3-pyruvate is coupled to a reaction forming a D-amino acidproduct, which D-amino acid can be recycled for use in the reactioncoupled to the R-MP reaction. This allows one to use non-stoichiometricamounts of amino acceptor in step one. In some embodiments of theinvention, the D-amino acid is D-alanine.

FIGS. 4 and 5 illustrate additional modifications of the pathway shownin FIG. 1, which modifications are directed to recycling the amino acidproduct formed by the reaction coupled with the L-tryptophan reactionwith the amino acid reactant of the reaction coupled to the MP tomonatin reaction.

Turning to FIG. 4, the recycling is accomplished providing an enzymethat can facilitate the conversion of an L-amino acid to a D-amino acidand vice versa. More specifically, where as is shown in FIG. 4, α-KGreacts to form L-glutamate when L-tryptophan reacts to formindole-3-pyruvate, a glutamate racemase (EC 5.1.1.3) or functionalequivalent can be provided that can facilitate the conversion ofL-glutamate to D-glutamate and vice versa. In such an instance, theL-glutamate formed alongside the production of indole-3-pyruvate isremoved by virtue of its conversion to D-glutamate, and the D-glutamateformed from the conversion of L-glutamate is then available as asubstrate for the reaction coupled with the MP to monatin reaction.Similarly, the α-KG formed in the reaction of D-glutamate is availableas a substrate for the reaction coupled to the L-tryptophan toindole-3-pyruvate reaction.

Non-limited examples of potential sources from which a glutamateracemase may be derived include Pediococcus pentosaceus, Bacilluspumilus, Lactobacillus fermenti, Lactobacillus brevis, E. coli, Aquifexpyrophilus, and Bacillus subtilis. More specifically (alsonon-limiting), the glutamate racemase may be expressed from a nucleicacid such as pediococcus pentaosaceus murl gene (Genbank Accession No.L22789), or Lactobacillus brevis glutamate racemase.

Where oxaloacetate reacts to form L-aspartate when L-tryptophan reactsto form indole-3-pyruvate, an aspartate racemase (EC 5.1.1.13) orfunctional equivalent can be provided to convert L-aspartate toD-aspartate. In such an instance, the L-aspartate alongside theproduction of indole-3-pyruvate is removed by virtue of its conversionto D-aspartate, and the D-aspartate formed from the conversion ofL-aspartate is then available to as a substrate for the reaction coupledto the MP to monatin reaction. Similarly, the oxaloacetate formed in thereaction of D-aspartate is available to act as a substrate for thereaction coupled to the L-tryptophan to indole-3-pyruvate reaction.

Non-limiting examples of suitable enzymes having aspartate racemaseactivity include ASPR-101 (BioCatalytics, Inc., Pasadena, Calif.) andhomologs or mutants of an amino acid racemase (EC 5.1.1.-) which arecapable of facilitating the conversion of L-aspartate to D-aspartate.

Non-limiting examples of potential sources from which aspartateracemases may be derived include: Desulfurococcus, Thermococcus, bivalvemollusk Scapharca brouhtonii, Acinetobacter, Agrobacterium,Archaeoglobus, Bacillus, Bordetella, Bradyrhizobium, Brevibacterium,Burkholderia, Campylobacter, Candida, Caulobacter, Clostridium,Desulfitobacterium, Desulfotalea, Enterococcus, Erwinia, Escherichia,Ferroplasma, Helicobacter, Klebsiella, Lactobacillus, Mannheimia,Medicago, Mesorhizobium, Methanococcus, Methanosarcina, Oceanobacillus,Oenococcus, Pediococcus, Polaribacter, Pseudomonas, Pyrococcus,Ralsonia, Shigella, Sinorhizobium, Salmonella, Sphingomonas,Streptococcus, Thermoanaerobacter, Vibrio, Wolinella, Xanthomonas,Xanthobacter, Yersinia and Zymomonas.

Where pyruvate reacts to form L-alanine when L-tryptophan reacts to formindole-3-pyruvate, an alanine racemase or functional equivalent can beprovided to convert L-alanine to D-alanine. In such an instance, theL-alanine formed alongside the production of indole-3-pyruvate isremoved by virtue of its conversion to D-alanine, and the D-alanineformed from the conversion of L-alanine is then available to act as asubstrate for the reaction coupled to the MP to monatin reaction.Similarly, the pyruvate formed in the reaction of D-alanine is availableto act as a substrate for the reaction couple with the L-tryptophan toindole-3-pyruvate reaction.

Non-limiting examples of suitable alanine racemases include A8936(Sigma-Aldrich, St. Louis, Mo.).

Non-limiting examples of potential sources from which the alanineracemase may be derived include: Brucella abortus, Streptococcusfaecalis Salmonella typhimurium, Escherichia coli, Bacillus subtilis,Bacillus stearothermophilus, Pseudomonas aeruginosa, Vibrio cholerae,Schizosaccaroyces pombe, Bacillus cereus and Lentinus edodes.

Examples 18 and 21 illustrate the use of the above racemases, theirimpact on increasing the ratio of the desired monatin product, andprovide potential sources for the racemase enzymes.

Turning to FIG. 5, a stereoinverting aminotransferase is used tofacilitate the reaction of R-MP to monatin. Although typically the R-MP(or S-MP) reaction to form R,R monatin (or S,R monatin) is coupled withthe reaction of a D-amino acid, a stereoinverting aminotransferase canfacilitate the coupled reactions of R-MP (or S-MP) to form R,R monatin(or S,R monatin) using an L-amino acid. In this way, the L-amino acidproduct of the L-tryptophan aminotransferase reaction can be used as asubstrate for the transamination of MP to monatin, and the product (i.e.oxaloacetate, pyruvate, and/or α-KG) of the reaction coupled to the MPto monatin reaction can be used as a starting material for the reactioncoupled to the L-tryptophan to indole-3-pyruvate reaction. Non-limitingexamples of stereoinverting aminotransferases that may be used includeD-phenylglycine aminotransferase (EC 2.6.1.72, also known asD-4-hydroxyphenylglycine aminotransferase) and D-methionineaminotransferase (EC 2.6.1.41, also known as D-met-aminotransferase andD-methionine-pyruvate aminotransferase). Non-limiting examples ofpotential sources from which the D-phenylglycine aminotransferase may bederived include Pseudomonas, such as Pseudomonas putida LW-4 andPseudomonas stutzeri ST-201. Non-limiting examples of potential sourcesfrom which the D-methionine aminotransferase may be derived includecauliflower and peanut.

Examples 19 and 20 together provide potential sources of stereoinvertingenzymes, and methods of making such enzymes. The examples also providescreening methods for identifying such enzymes. It is also contemplatedthat such enzymes may be evolved from stereoinverting enzymes known orfound in nature. As a non-limiting example, the stereoinvertingaminotransferase may be a homolog or mutant of a D-amino acidaminotransferase or a homolog or mutant of an amino acid racemase (EC5.1.1.-).

FIGS. 6-8 also illustrate modifications to the pathway of FIG. 1. Thepathways illustrated in FIGS. 6-8 provide methods to push equilibriumreactions forward by removing byproduct of the tryptophan reaction andin some cases providing substrate for the MP reaction.

Turning to FIG. 6, the pathway shown removes the L-amino acid product ofthe reaction coupled to the tryptophan reaction by converting it to adifferent L-amino acid, and then provides a substrate for reactioncoupled to the MP reaction by converting the newly formed L-amino acidto a D-amino acid. Specifically, L-tryptophan is shown to reactalongside oxaloacetate to form indole-3-pyruvate and L-aspartate. Anaspartate 4-decarboxylase (EC 4.1.1.12) or functional equivalent is usedto facilitate the conversion of L-aspartate to L-alanine and carbondioxide, and an enzyme with alanine racemase activity is used tofacilitate the conversion of L-alanine to D-alanine, which D-alanine canserve as an amino donor for the conversion of R-MP to monatin.

Turning to FIG. 7, the pathway shown illustrates additional methods forremoving the L-amino acid product of the reaction coupled to thetryptophan reaction. Embodiments as presented in the figure produce abyproduct(s) that is unavailable to react in the reverse direction, forexample due to volatility (such as carbon dioxide) or by spontaneousconversion to an unreactive endproduct. An example of such an approachincludes where α-KG reacts alongside L-tryptophan to produceL-glutamate, a glutamate decarboxylase (EC 4.1.1.15) or functionalequivalent can be provided which can facilitate the conversion ofL-glutamate to 4-aminobutanoate (with carbon dioxide as a byproduct).Non-limiting examples of potential sources from which the L-glutamatedecarboxylase may be derived include: Clostridium perfringens, C.welchii, or E. coli.

Another example of such an approach for moving the tryptophan reactionforward includes where oxaloacetate reacts alongside L-tryptophan, anaspartate decarboxylase (EC 4.1.1.11) or functional equivalent can beprovided to facilitate the conversion of L-aspartate to β-alanine (withcarbon dioxide as a byproduct).

Turning to FIG. 8, the pathway shown illustrates yet additional methodsfor removing the L-amino acid product of the reaction coupled to thetryptophan reaction and providing a substrate for the reaction coupledto the MP reaction. Specifically, where α-KG reacts alongsideL-tryptophan to form L-glutamate, an enzyme with L-alanineaminotransferase activity and pyruvate can be provided, wherein theL-alanine aminotransferase enzyme facilitates the reaction of pyruvateand L-glutamate to form L-alanine. An alanine racemase or functionalequivalent can also be provided in order to facilitate the conversion ofthe L-alanine to D-alanine, which D-alanine can be used as a substratealong with MP to form monatin and pyruvate. See Examples 18 and 21.

Biosynthetic Pathways to Produce R,R and Other Stereoisomers of MonatinDerivatives

The methods of the described invention include using the polypeptideswith aldolase activity described herein may be used to facilitate thereaction between a substituted indole-3-pyruvate and a C3 carbon source.

Enzymes useful for the facilitating a reaction between a substitutedindole-3-pyruvate and a C3 carbon source include one or morepolypeptides with aldolase activity of any of SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ IDNO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ IDNO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ IDNO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ IDNO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124,SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ IDNO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152,SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ IDNO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180,SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ IDNO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208,SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ IDNO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236,SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ IDNO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQID NO:256, SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264,SEQ ID NO:266, SEQ ID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ IDNO:274, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQID NO:284, SEQ ID NO:286, SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:292,SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ IDNO:302, SEQ ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320,SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ IDNO:330, SEQ ID NO:332, or SEQ ID NO:334, or fragments or subsequencesthereof having aldolase activity.

In one embodiment, one or more polypeptides with HMG aldolase activityof any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ IDNO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ IDNO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ IDNO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118,SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ IDNO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146,SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ IDNO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174,SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ IDNO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202,SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ IDNO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230,SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ IDNO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258,SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQ IDNO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQ ID NO:276, SEQID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:284, SEQ ID NO:286,SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:292, SEQ ID NO:294, SEQ IDNO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ ID NO:302, SEQ ID NO:304 orfragments or subsequences thereof having aldolase activity may be usefulin facilitating a reaction between a substituted indole-3-pyruvate and aC3 carbon source.

In another embodiment, one or more polypeptides with KHG aldolaseactivity of any of SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ IDNO:312, SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQID NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330,SEQ ID NO:332, or SEQ ID NO:334 or fragments or subsequences thereofhaving aldolase activity may be useful in facilitating a reactionbetween a substituted indole-3-pyruvate and a C3 carbon source.

Alternatively, one or more polypeptides with aldolase activity encodedby a nucleic acid sequence having at least about 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to anucleic acid in accordance with the invention, including SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ IDNO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ IDNO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ IDNO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ IDNO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ IDNO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ IDNO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121,SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ IDNO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149,SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ IDNO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177,SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ IDNO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205,SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ IDNO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233,SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ IDNO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261,SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269, SEQ IDNO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQID NO:281, SEQ ID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQ ID NO:289,SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ IDNO:299, SEQ ID NO:301, SEQ ID NO:303, SEQ ID NO:305, SEQ ID NO:307, SEQID NO:309, SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317,SEQ ID NO:319, SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325, SEQ IDNO:327, SEQ ID NO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ ID NO:335, SEQID NO:336, SEQ ID NO:337, and SEQ ID NO:338 over a region of at leastabout 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600,1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2200, 2250,2300, 2350, 2400, 2450, 2500, or more residues may be useful infacilitating a reaction between a substituted indole-3-pyruvate and a C3carbon source.

In one embodiment of the invention, one or more polypeptides with HMGaldolase activity encoded by a nucleic acid sequence having at leastabout 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)sequence identity to a nucleic acid in accordance with the invention,including SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117,SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ IDNO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145,SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ IDNO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173,SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ IDNO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201,SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ IDNO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229,SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ IDNO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257,SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ IDNO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:285,SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ IDNO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQID NO:305 over a region of at least about 10, 15, 20, 25, 30, 35, 40,45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900,1950, 2000, 2050, 2100, 2200, 2250, 2300, 2350, 2400, 2450, 2500, ormore residues may be useful in facilitating a reaction between asubstituted indole-3-pyruvate and a C3 carbon source.

In another embodiment of the invention, one or more polypeptides withKHG aldolase activity encoded by a nucleic acid sequence having at leastabout 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)sequence identity to a nucleic acid in accordance with the invention,including SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ ID NO:313,SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321, SEQ IDNO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQID NO:333, SEQ ID NO:335, SEQ ID NO:336, SEQ ID NO:337, and SEQ IDNO:338 over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45,50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950,2000, 2050, 2100, 2200, 2250, 2300, 2350, 2400, 2450, 2500, or moreresidues may be useful in facilitating a reaction between a substitutedindole-3-pyruvate and a C3 carbon source.

One or more polypeptides with aldolase activity encoded by a nucleicacid sequence that hybridizes under stringent condition to a nucleicacid of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19,SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29,SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39,SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49,SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59,SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69,SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79,SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89,SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99,SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ IDNO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127,SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ IDNO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155,SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ IDNO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183,SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ IDNO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211,SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ IDNO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239,SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ IDNO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267,SEQ ID NO:269, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ IDNO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:285, SEQID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295,SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQ IDNO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ ID NO:313, SEQID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321, SEQ ID NO:323,SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQ IDNO:333, SEQ ID NO:335, SEQ ID NO:336, SEQ ID NO:337, and SEQ ID NO:338may be useful in facilitating a reaction between a substitutedindole-3-pyruvate and a C3 carbon source.

In one embodiment of the invention, one or more polypeptides with HMGaldolase activity encoded by a nucleic acid sequence that hybridizesunder stringent condition to a nucleic acid of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ IDNO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151,SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ IDNO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179,SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ IDNO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207,SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ IDNO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235,SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ IDNO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263,SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ IDNO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291,SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ IDNO:301, SEQ ID NO:303, SEQ ID NO:305 may be useful in facilitating thereaction between the substituted indole-3-pyruvate and the C3 carbonsource.

In another embodiment of the invention, one or more polypeptides withKHG aldolase activity encoded by a nucleic acid sequence that hybridizesunder stringent condition to a nucleic acid of SEQ ID NO:307, SEQ IDNO:309, SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQID NO:319, SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327,SEQ ID NO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ ID NO:335, SEQ IDNO:336, SEQ ID NO:337, and SEQ ID NO:338 may be useful in facilitating areaction between a substituted indole-3-pyruvate and a C3 carbon source.

In one embodiment, the substituent group of the substitutedindole-3-pyruvate is a halogen atom attached to any carbon atom of theindole ring. In another embodiment, the substituent group is a chlorineatom attached to any carbon of the indole ring. In yet anotherembodiment, the monatin derivative is4-hydroxy-4-(6-methylindole-3-ylmethyl)glutamic acid.

Polypeptides having aldolase activity, and in accordance with someembodiments of the invention, may be used in a multi-step pathway inwhich one or more step is a chemical synthesis reaction. For example, insome embodiments, one or more polypeptides having aldolase activity canfacilitate a reaction between pyruvate and indole-3-pyruvate to yieldmonatin precursor. The monatin precursor can then be purified. Areductive amination reaction of the monatin precursor can then beutilized to yield monatin.

Polypeptides having aldolase activity, and in accordance with someembodiments of the invention, as well as the other enzymes used in theprocess for producing monatin and monatin derivatives may be used inpure, crude, isolated, or ammonium sulfate suspension form.

Polypeptides having aldolase activity, and in accordance with someembodiments of the invention, may be optimized using stabilizing agents,including dithiothreitol (“DTT”) and β-mercaptoethanol.

Monatin or monatin derivative that is produced utilizing one or more ofthe polypeptides disclosed herein, is generally at least about 50 toabout 99% R,R-monatin or R,R-monatin derivative, by weight of the totalmonatin or monatin derivative produced. In other embodiments, themonatin or monatin derivative produced utilizing one or more of thepolypeptides disclosed herein, is greater than 60% R,R-monatin orR,R-monatin derivative, by weight of the total monatin produced; forexample, the R,R-monatin or R,R-monatin derivative is 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the totalmonatin or monatin derivative produced Alternatively, various amounts oftwo or more preparations of monatin or monatin derivative can becombined so as to result in a preparation that is a desired percentageof R,R-monatin or R,R-monatin derivative. For example, a monatinpreparation that is 60% R,R-monatin can be combined with a monatinpreparation that is 90% R,R-monatin; if equal amounts of 60% and 90%R,R-monatin preparations are combined, the resulting monatin preparationwould be 75% R,R-monatin.

The monatin or monatin derivative, or an intermediate (including monatinprecursor), produced utilizing one or more of the polypeptides disclosedherein, may be purified from the components of the reaction. In oneembodiment, the monatin, monatin derivative or intermediate, such asmonatin precursor, may be purified simply by removing the substance thatis to be purified from the enzyme preparation in which it wassynthesized.

In other embodiments, the intermediate, monatin precursor, monatin ormonatin derivative is purified from a preparation in which it wassynthesized so that the resulting “purified” composition or preparationis at least about 5-60% monatin by weight of total organic compounds. Inanother embodiment, the monatin, monatin derivative or intermediate,such as monatin precursor, may be purified to a degree of purity of atleast about 70%, 80%, 90%, 95% or 99% by weight of total organiccompounds. The monatin, monatin derivative or the intermediate(including monatin precursor), produced utilizing one or more of thepolypeptides disclosed herein, may be purified from the components ofthe reaction by any method known to a person of ordinary skill in theart. Optimally, the purified monatin or intermediate may be repeatedlyrecrystallized until the desired degree of purity is achieved.

The following examples are offered to illustrate, but not to limit theclaimed invention.

EXAMPLES Example 1 Detection of Monatin, Monatin Precursor, Tryptophan,Alanine, Aspartate, and Glutamate

This example describes methods used to detect the presence of monatin,monatin precursor (“MP”), tryptophan, aspartate, alanine, and glutamate.It also describes a method for the separation and detection of the fourstereoisomers of monatin.

LC/MS/MS Multiple Reaction Monitoring (“MRM”) Analysis of Monatin andTryptophan

Analyses of mixtures for monatin and tryptophan derived from in vitro orin vivo biochemical reactions were performed using a Waters/Micromassliquid chromatography-tandem mass spectrometry (LC/MS/MS) instrumentincluding a Waters 2795 liquid chromatograph with a Waters 996Photo-Diode Array (PDA) absorbance monitor placed in series between thechromatograph and a Micromass Quattro Ultima triple quadrupole massspectrometer. LC separations were made using an Xterra MS C₈reversed-phase chromatography column, 2.1 mm×250 mm at 40° C. The LCmobile phase consisted of A) water containing either (i) 0.05% (v/v)trifluoracetic acid or (ii) 0.3% formic acid and 10 mM ammonium formateand B) methanol containing either (i) 0.05% (v/v) trifluoracetic acid or(ii) 0.3% formic acid and 10 mM ammonium formate.

If the LC mobile phase consisted of A) water containing 0.05% (v/v)trifluoracetic acid and B) methanol containing 0.05% (v/v)trifluoracetic acid, gradient elution was linear from 5% B to 35% B, 0-4minutes, linear from 35% B to 60% B, 4-6.5 minutes, linear from 60% B to90% B, 6.5-7 minutes, isocratic at 90% B 7-11 minutes, linear from 90% Bto 95% B, 11-12 minutes, linear from 95% B to 5% B, 12-13 minutes, witha 2 minute re-equilibration period between runs. The flow rate was 0.25mL/min, and PDA absorbance was monitored from 200 nm to 400 nm Allparameters of the ESI-MS were optimized and selected based on generationof protonated molecular ions ([M+H]+) of the analytes of interest, andproduction of characteristic fragment ions. The following instrumentalparameters were used for LC/MS/MS Multiple Reaction Monitoring (MRM)analysis of monatin and tryptophan: Capillary: 3.5 kV; Cone: 40 V; Hex1: 20 V; Aperture: 0 V; Hex 2: 0 V; Source temperature: 100° C.;Desolvation temperature: 350° C.; Desolvation gas: 500 L/h; Cone gas: 50L/h; Low mass resolution (Q1): 12.0; High mass resolution (Q1): 12.0;Ion energy: 0.2; Entrance: −5 V; Collision Energy: 8; Exit: 1V; Low massresolution (Q2): 15; High mass resolution (Q2): 15; Ion energy (Q2):3.5; Multiplier: 650. Five monatin—specific parent—to daughter MRMtransitions are used to specifically detect monatin in in vitro and invivo reactions. The transitions monitored are 293.1 to 158.3, 293.1 to168.2, 293.1 to 211.2, 293.1 to 230.2, and 293.1 to 257.2. Tryptophan ismonitored with the MRM transition 204.7 to 146.4. For internal standardquantification of monatin and tryptophan, four calibration standardscontaining four different ratios of each analyte to d5-tryptophan andd5-monatin, are analyzed. These data are subjected to a linear leastsquares analysis to form a calibration curve for monatin and tryptophan.To each sample is added a fixed amount of d5-tryptophan and d5-monatin(d5-monatin was synthesized from d5-tryptophan according to the methodsfrom WO03/091396 A2), and the response ratios (monatin/d5-monatin;tryptophan/d5-tryptophan) used in conjunction with the calibrationcurves described above to calculate the amount of each analyte in themixtures.

If the LC mobile phase was A) water containing 0.3% formic acid and 10mM ammonium formate and B) methanol containing 0.3% formic acid and 10mM ammonium formate, the gradient elution was linear from 5% B to 45% B,0-8.5 minutes, linear from 45% B to 90% B, 8.5-9 minutes, isocratic from90% B to 90% B, 9-12.5 minutes, linear from 95% B to 5% B, 12.5-13minutes, with a 4 minute re-equilibration period between runs. The flowrate was 0.27 mL/min, and PDA absorbance was monitored from 210 nm to400 nm. All parameters of the ESI-MS were optimized and selected basedon generation of protonated molecular ions ([M+H]+) of the analytes ofinterest, and production of characteristic fragment ions. Theinstrumental parameters used for this secondary mobile phase are thesame as above. Four monatin—specific parent—to daughter MRM transitionsand one tryptophan specific parent to daughter transition are used tospecifically detect monatin and tryptophan in in vitro and in vivoreactions. The transitions monitored are 293.1 to 158.0, 293.1 to 168.0,293.1 to 211.5, and 293.1 to 257.0. Tryptophan is monitored with the MRMtransition 205.2 to 146.1. For internal standard quantification ofmonatin and tryptophan, four calibration standards containing fourdifferent ratios of each analyte to d5-tryptophan and d5-monatin, areanalyzed. These data are subjected to a linear least squares analysis toform a calibration curve for monatin and tryptophan. To each sample isadded a fixed amount of d5-tryptophan and d5-monatin (d5-monatin wassynthesized from d5-tryptophan according to the methods from WO03/091396A2), and the response ratios (monatin/d5-monatin;tryptophan/d5-tryptophan) in conjunction with the calibration curvesdescribed above are used to calculate the amount of each analyte in themixtures. Parent to daughter mass transitions monitored ford5-tryptophan and d5-monatin are 210.2 to 151.1, and 298.1 to 172.0respectively.

Accurate Mass Measurement of Monatin

High resolution MS analysis was carried out using an AppliedBiosystems-Perkin Elmer Q-Star hybrid quadrupole/time-of-flight massspectrometer. The measured mass for protonated monatin used tryptophanas an internal mass calibration standard. The calculated mass ofprotonated monatin, based on the elemental composition C14H17N2O5 is293.1137. Monatin produced using the biocatalytic process described inExamples 2 and 3 showed a measured mass of 293.1144. This is a massmeasurement error of less than 2 parts per million (“ppm”), providingconclusive evidence of the elemental composition of monatin producedenzymatically.

Chiral LC/MS/MS (“MRM”) Measurement of Monatin

Determination of the stereoisomer distribution of monatin in in vitroand in vivo reactions was accomplished by derivitization with1-fluoro-2-4-dinitrophenyl-5-L-alanine amide (“FDAA”), followed byreversed-phase LC/MS/MS MRM measurement.

Derivatization of Monatin with FDAA

To 50 μL of sample or standard and 10 μL of internal standard was addedeither 100 μL or 200 μL of a 1% solution of FDAA in acetone. Twenty orforty μL, respectively, of 1.0 M sodium bicarbonate was added, and themixture incubated for 1 h at 40° C. with occasional mixing. The samplewas removed and cooled, and neutralized with 20 μL of 2.0 M HCl (moreHCl may be required to effect neutralization of a buffered biologicalmixture). After degassing was complete, samples were ready for analysisby LC/MS/MS.

LC/MS/MS Multiple Reaction Monitoring for the Determination of theStereoisomer Distribution of Monatin in in vitro and in vivo Reactions

Analyses were performed using the LC/MS/MS instrumentation describedabove. LC separations capable of separating all four stereoisomers ofmonatin (specifically FDAA-monatin) were performed on a Phenomenex Luna2.0×250 mm (3 μm) C18 (2) reversed phase chromatography column at 40° C.The LC mobile phase consisted of A) water containing 0.05% (mass/volume)ammonium acetate and B) acetonitrile. The elution was isocratic at 13%B, 0-2 minutes, linear from 13% B to 30% B, 2-15 minutes, linear from30% B to 80% B, 15-16 minutes, isocratic at 80% B 16-21 minutes, andlinear from 80% B to 13% B, 21-22 minutes, with an 8 minutere-equilibration period between runs. The flow rate was 0.23 mL/min, andPDA absorbance was monitored from 200 nm to 400 nm. All parameters ofthe ESI-MS were optimized and selected based on generation ofdeprotonated molecular ions ([M−H]−) of FDAA-monatin, and production ofcharacteristic fragment ions.

The following instrumental parameters were used for LC/MS analysis ofmonatin in the negative ion ESI/MS mode: Capillary: 2.0 kV; Cone: 25 V;Hex 1: 10 V; Aperture: 0 V; Hex 2: 0 V; Source temperature: 100° C.;Desolvation temperature: 350° C.; Desolvation gas: 500 L/h; Cone gas: 50L/h; Low mass resolution (Q1): 12.0; High mass resolution (Q1): 12.0;Ion energy: 0.2; Entrance: −5V; Collision Energy: 20; Exit: 1V; Low massresolution (Q2): 12; High mass resolution (Q2): 12; Ion energy (Q2):3.0; Multiplier: 650. Three FDAA-monatin—specific parent—to daughtertransitions are used to specifically detect FDAA-monatin in in vitro andin vivo reactions. The transitions monitored for monatin are 543.2 to268.1, 543.2 to 499.3, and 543.2 to 525.3. Monatin internal standardderivative mass transition monitored was 548.2 to 530.3. Identificationof FDAA-monatin stereoisomers is based on chromatographic retention timeas compared to purified synthetic monatin stereoisomers, and massspectral data. An internal standard is used to monitor the progress ofthe reaction and for confirmation of retention time of the S,Sstereoisomer.

Liquid Chromatography-Post Column Fluorescence Detection of Amino AcidsIncluding Glutamate and Alanine

Liquid chromatography with post-column fluorescence detection (LC/OPA)for the determination of glutamate and alanine in in vitro and in vivoreactions was performed on a Waters 2690 LC system or equivalentcombined with a Waters 474 scanning fluorescence detector, and a Waterspost-column reaction module. Semi-quantitative analyses of monatin andtryptophan were also performed using this method. LC separations wereperformed on an Interaction-Sodium loaded ion exchange column at 60° C.Mobile phase A was Pickering Na 328 buffer (Pickering Laboratories,Inc.; Mountain View, Calif.). Mobile phase B was Pickering Na 740buffer. The gradient elution was from 0% B to 100% B, 0-20 minutes,isocratic at 100% B, 20-36 minutes, and linear from 100% B to 0% B,36-37 minutes, with at least a 5 minute re-equilibration period betweenruns, depending on sample matrix. The flow rate for the mobile phase was0.5 mL/min. The flow rate for the OPA post-column derivatizationsolution was 0.5 mL/min. The fluorescence detector settings were EX338-340 nm and Em 420-425 nm. Norleucine was employed as an internalstandard for the analysis. Identification of amino acids was based onchromatographic retention time data for purified standards.

Detection of L- and D-Amino Acids by LC/MS/MS

Samples containing a mixture of L- and D-amino acids such as lysine,alanine, methionine, tyrosine, leucine, phenylalanine, tryptophan,glutamate, and aspartate from biochemical reaction experiments werefirst treated with formic acid to denature protein. The sample was thencentrifuged and filtered through a 0.45 μm nylon syringe filter prior toLC/MS/MS analysis. Identification of L- and D-amino acids was based onretention time and mass selective detection. LC separation wasaccomplished by using Waters 2690 liquid chromatography system and anASTEC 2.1 mm×250 mm Chirobiotic TAG chromatography column with columntemperature set at 45o C. LC mobile phase A and B were 0.25% acetic acidand 0.25% acetic acid in methanol, respectively. Isocratic elution wasused for all methods to separate the L and D isomers. Lysine was elutedusing 80% mobile phase A, and 20% B. Glutamate, alanine, and methioninewere separated with elution of 60% mobile phase A and 40% B and a flowrate of 0.25 mL/min Aspartate, tryptophan, tyrosine, leucine, andphenylalanine were separated isomerically with 30% mobile phase A and70% B with a flow rate of 0.3 mL/min for all but phenylalanine, whichwas run at a flow rate of 0.25 mL/min

The detection system for analysis of L- and D-amino acids included aWaters 996 Photo-Diode Array (PDA) detector and a Micromass QuattroUltima triple quadrupole mass spectrometer. The PDA, scanning from 195to 350 nm, was placed in series between the chromatography system andthe mass spectrometer. Parameters for the Micromass Quattro Ultimatriple quadrupole mass spectrometer operating in positive electrosprayionization mode (+ESI) were set as the following: Capillary: 3.0 kV;Cone: 20 V; Hex 1: 15 V; Aperture: 1 V; Hex 2: 0 V; Source temperature:100° C.; Desolvation temperature: 350° C.; Desolvation gas: 530 L/h;Cone gas: 30 L/h; Low mass Q1 resolution: 12.5; High mass Q1 resolution:12.5; Ion energy 1: 0.2; Entrance: −5; Collision: 8; Exit 1: 10; Lowmass Q2 resolution: 12.5; High mass Q2 resolution: 12.5; Ion energy 2:0.5; Multiplier: 650 V. MS/MS experiments with Multiple ReactionMonitoring (MRM) mode were set up to selectively monitor reactiontransitions of 147.8 to 84.2 and 147.8 to 102.1 for glutamate, 134.00 to74.30, and 134.00 to 88.2 for aspartate, 147.3 to 85.0 for lysine, 150.3to 104.8 for methionine, 182.3 to 137.0 for tyrosine, 132.3 to 87.0 forleucine, and 166.3 to 121.0 for phenylalanine. In the case where twotransitions are listed, the latter transitions were used forquantification. For tryptophan, MS/MS experiments with Multiple ReactionMonitoring (MRM) mode were set up to selectively monitor reactiontransitions of 205.2 to 118.2, 205.2 to 146.1, and 205.2 to 188.2, andthe transition from 212.1 to 151.1 for d8-DL tryptophan. Tryptophanquantification was achieved by determining the ratio of analyte responseof transition 205.2 to 146.1 to that of the internal standard, d8-D,Ltryptophan. Alternatively, quantification of tryptophan, glutamate, andaspartic acids were based off signal responses of m/z=146.5, m/z=102.1,and m/z=88.2, respectively.

Production of Monatin and Monatin Precursor (“MP”) for Standards and forAssays

Production of Monatin

A racemic mixture of R,R and S,S monatin was synthetically produced asdescribed in U.S. Pat. No. 5,128,482.

The R,R and S,S monatin were separated by a derivatization andhydrolysis step. Briefly, the monatin racemic mixture was esterified,the free amino group was blocked with Cbz, a lactone was formed, and theS,S lactone was selectively hydrolyzed using an immobilized proteaseenzyme. The monatin can also be separated as described in Bassoli, A. etal., Eur. J. Org. Chem., 8:1652-1658, (2005).

MP Production

R-MP was produced by the transamination of R,R monatin using AT-103broad range D-aminotransferase (BioCatalytics, Pasadena, Calif.) in 0.1Mpotassium phosphate buffer, using sodium pyruvate as the amino acceptor.S-MP was produced by the transamination of S,S monatin using AT-102L-aminotransferase (BioCatalytics, Pasadena, Calif.) in 0.1M potassiumphosphate buffer, using sodium pyruvate as the amino acceptor. Bothreactions were carried out at 30° C. and at a pH of approximately8.0-8.3, for approximately 20 hours. Both compounds were purified usingpreparative scale HPLC with a Rohm and Haas (Philadelphia, Pa.)hydrophobic resin (XADTM1600), eluting in water. Samples containinggreater than 90% purity monatin precursor were collected andfreeze-dried.

Example 2 Detection of Monatin Precursor

This example describes methods used for the separation and detection ofthe two enantiomers of monatin precursor.

Non-chiral Method for Detection of Monatin Precursor

Reaction samples from 96-well plates were injected onto an AgilentZorbax RX-C18, 3.5 um, 3.0×150 mm column using a CTCPal auto-sampler(LEAP Technologies, Carrboro, N.C.). Products were separated using aH2O/ACN (0.1% Formic acid) gradient:

Time: 0.00 min 5% B Time: 4.00 min 100% B  Time: 5.00 min 100% B  Time:5.10 min 5% B Time: 6.50 min 5% B

The gradient was provided by LC-10ADvp pumps (Shimadzu, Kyoto, Japan) at0.8 mL/min. Products were detected using API4000 TurboIon-Spraytriple-quad mass spectrometer (Applied Biosystems, Foster City, Calif.).Ion spray and Multiple-ion monitoring were performed for the analytes ofinterest in the negative ion mode, and each analysis lasted 6.5 minutes.

Pyruvate=87.11 [M−H+]—

Indole-3-pyruvate=202.1 [M−H+]—

Product=290.0 [M−H+]— Chiral CE Analysis of R & S Monatin Precursors

A P/ACE™ MDQ capillary electrophoresis instrument (Beckman Coulter,Fullerton, Calif.) was used. The Chiral Development kit was used andincludes small amounts of several chiral selectors, necessary buffersand 2 capillaries (Beckman Coulter, Fullerton, Calif.). Alternatively,for the MP assay only, the following reagents and other supplies can beobtained separately from Beckman Coulter (Fullerton, Calif.) orelsewhere:

Coated capillary N—CHO; 50 um ID, 65 cm total length or fused silicacapillary.

25 mM phosphate buffer, pH 5

25 mg hydroxypropyl-β-cyclodextrin

Capillary conditioning solution, 10 mL (alternatively, can use 0.5%polyethylene oxide solution, M_(w) 600,000 or 300,000 Daltons)

Capillary Electrophoresis (“CE”) Analysis

A neutral coated capillary, 50 um ID, 60 cm (50 cm to detection) or 30(20) cm was used along with DAD detection (or simple UV) at 214 nm. Theseparation capillary was thermostated at 15° C., samples at 4° C. Theseparation buffer was 20 mM hydroxypropyl-β-cycylodextrin, 25 mMphosphate, pH 5. Sample injection was typically 0.5 psi, 5 s. Separationwas at 500 V/cm, reversed polarity (15 kV for 30 cm capillary, 30 kV for60 cm). Typical current used during separation was −28 μA. Typicalmigration times for MP peaks were around 3.5 minutes (20 cm effectivelength) or 8 minutes (50 cm)

An optional capillary cleaning/washing/conditioning step, prior tosample runs used H₂O 4 minutes, 0.1M HCl 1 minutes, H₂O 1.5 minutes,capillary conditioning solution 4 minutes, H₂O, 1 minute, separationbuffer 4 minutes.

A summary of the run method was: separation buffer rinse 1-2 minutes,sample injection 5 s at 0.5 psi, separation 5-10 minutes at reversedvoltage polarity 15 or 30 kV, depending on capillary length.

Example 3 General Assay for Pyruvate Aldolases

An exemplary method to measure the activity of different pyruvatealdolases uses a general substrate, 4-Carboxy-4-hydroxy-2-oxoadipate(CHA). The CHA assay was adapted from literature assays (eg. See E. E.Dekker & R. P. Kitson, J. Biol. Chem. 267, 10507-10514, 1992). A typicalassay comprised 50 mM sodium phosphate pH 7.5, 1 mM MgCl₂, 1 mM CHA, 10μg/ml D-lactate dehydrogenase (LDH) from Lactobacillus leichmanii(Sigma-Aldrich, St. Louis, Mo.), 0.5 mM NADH. The assay was started byadding enzyme (typically between 1 to 5 mL). Liberation of pyruvate,coupled to the formation of NAD⁺ was monitored continuously in aspectrophotometer at 340 nm.

The CHA was synthesized according to the procedure described in Tack, B.F. Chapman, P. J., and S. Dagley. J. Biol. Chem. 247 6438-6443 (1972).

A unit of enzyme activity, such as pyruvate aldolase, such as HMG and/orKHG aldolase enzyme, is defined as the amount that liberates sufficientpyruvate to lower the absorbance at 340 nm by 1 OD per minute.

Example 4 Discovery of Novel Keto-Hydroxy-Glutarate (KHG) andHydroxy-Methyl-Keto-Glutarate (HMG) Aldolases from Diversa EnvironmentalLibraries

Over 150 unique HMG aldolases and 15 KHG aldolases were discovered byscreening Divers a DNA libraries. These aldolase genes were sequencedand subcloned into a suitable expression vector. This vector was thentransformed into a suitable expression host for production of sufficientamounts of the aldolase for enzyme characterization. A selected set ofaldolases were tested for the activity on CHA and also for the formationof monatin precursor (MP). All the enzymes discovered and described inthis patent have potential for use in other carbon-carbon bond formingreactions between an alpha-keto acid acceptor and pyruvate or pyruvatederivative donor as exemplified in the general reaction scheme below.

Example 5 Characterization of Selected Aldolases

Selected aldolases were characterized with respect to their ability tocatalyze the conversion of indole-3-pyruvate and pyruvate to monatinprecursor (MP) as shown in the following scheme:

FIGS. 13 and 14 show the activities of 58 different aldolases in theformation of MP as measured by LC/MS/MS.

Aldol reactions were performed with 20 mM indole-3-pyruvate (“I3P”), 50mM pyruvate, 100 mM sodium phosphate pH 7, 1 mM MgCl₂, 100 μg/mLaldolase. Reactions were incubated at room temperature in the dark.Aliquots (30 μL) were removed at various times and reactions werestopped by storing the samples on ice. A portion of each aliquot wassubmitted for CE analysis while the remaining portion was diluted 1:1000in 50% acetonitrile and submitted for LC/MS analysis.

TABLE 2 Enantioselectivity of different aldolases for the formation ofMP from I3P and pyruvate as determined by chiral CE % R-MP CHA (U/mg)(23 hr time (based on total Relative Aldolase point) protein in lysate)Expression SEQ ID NO: 28 (encoded by SEQ ID NO: 27) 98+ 31.8 ### SEQ IDNO: 116 (encoded by SEQ ID NO: 115) 95 304 ## SEQ ID NO: 76 (encoded bySEQ ID NO: 75) 50 424 ### SEQ ID NO: 298 (encoded by SEQ ID NO: 297) 98+388 ## SEQ ID NO: 44 (encoded by SEQ ID NO: 43) 70 332 ## SEQ ID NO: 54(encoded by SEQ ID NO: 53) 98+ 95 # SEQ ID NO: 148 (encoded by SEQ IDNO: 147) 90 200 ## SEQ ID NO: 46 (encoded by SEQ ID NO: 45) 70 174 ##SEQ ID NO: 134 (encoded by SEQ ID NO: 133) 90 576 ### SEQ ID NO: 142(encoded by SEQ ID NO: 141) 98+ 55 # SEQ ID NO: 122 (encoded by SEQ IDNO: 121) 98+ 38 ## SEQ ID NO: 74 (encoded by SEQ ID NO: 73) 80 484 ###SEQ ID NO: 64 (encoded by SEQ ID NO: 63) 95 38 # SEQ ID NO: 108 (encodedby SEQ ID NO: 107) 98+ 40 # SEQ ID NO: 96 (encoded by SEQ ID NO: 95) 98+not detected # SEQ ID NO: 126 (encoded by SEQ ID NO: 125) 95 124 ## SEQID NO: 80 (encoded by SEQ ID NO: 79) 98+ not detected # SEQ ID NO: 36(encoded by SEQ ID NO: 35) 98+ 80 ## SEQ ID NO: 62 (encoded by SEQ IDNO: 61) 98+ not detected # SEQ ID NO: 112 (encoded by SEQ ID NO: 111)98+ not detected # SEQ ID NO: 130 (encoded by SEQ ID NO: 129) 98+ 38 ##SEQ ID NO: 94 (encoded by SEQ ID NO: 93) 98+ 47 ## SEQ ID NO: 102(encoded by SEQ ID NO: 101) not detected not detected # SEQ ID NO: 58(encoded by SEQ ID NO: 57) 98+ 59 ## SEQ ID NO: 88 (encoded by SEQ IDNO: 87) 50 510 ### SEQ ID NO: 50 (encoded by SEQ ID NO: 49) 98+ 144 ##SEQ ID NO: 106 (encoded by SEQ ID NO: 105) 98+ not detected ## SEQ IDNO: 40 (encoded by SEQ ID NO: 39) 40 406 ### SEQ ID NO: 42 (encoded bySEQ ID NO: 41) 98+ 92 ## SEQ ID NO: 278 (encoded by SEQ ID NO: 277) 952.0 # SEQ ID NO: 162 (encoded by SEQ ID NO: 161) 95 11.8 # SEQ ID NO:276 (encoded by SEQ ID NO: 275) 98+ 100.4 #### SEQ ID NO: 178 (encodedby SEQ ID NO: 177) 95 38.8 # SEQ ID NO: 202 (encoded by SEQ ID NO: 201)not detected not detected # SEQ ID NO: 166 (encoded by SEQ ID NO: 165)98+ 85.5 ## SEQ ID NO: 218 (encoded by SEQ ID NO: 217) 95 49.1 ## SEQ IDNO: 224 (encoded by SEQ ID NO: 223) 98+ 23.2 # SEQ ID NO: 226 (encodedby SEQ ID NO: 225) 98+ 128.3 # SEQ ID NO: 244 (encoded by SEQ ID NO:243) 98+ 40.4 # SEQ ID NO: 250 (encoded by SEQ ID NO: 249) 95 6.0 # SEQID NO: 252 (encoded by SEQ ID NO: 251) 95 20.2 ## SEQ ID NO: 264(encoded by SEQ ID NO: 263) 95 9.9 ## SEQ ID NO: 268 (encoded by SEQ IDNO: 267) 95 2.0 # SEQ ID NO: 272 (encoded by SEQ ID NO: 271) 95 6.7 #SEQ ID NO: 184 (encoded by SEQ ID NO: 183) 95 not detected # SEQ ID NO:282 (encoded by SEQ ID NO: 281) 95 36.7 ### SEQ ID NO: 186 (encoded bySEQ ID NO: 185) 95 4.2 # SEQ ID NO: 192 (encoded by SEQ ID NO: 191) 9511.9 # SEQ ID NO: 200 (encoded by SEQ ID NO: 199) 95 17.9 ## SEQ ID NO:280 (encoded by SEQ ID NO: 279) 50 not detected # SEQ ID NO: 284(encoded by SEQ ID NO: 283) 90 2.2 # SEQ ID NO: 172 (encoded by SEQ IDNO: 171) 95 8.4 # SEQ ID NO: 180 (encoded by SEQ ID NO: 179) 98+ 61.0### SEQ ID NO: 168 (encoded by SEQ ID NO: 167) 98+ 9.3 # SEQ ID NO: 228(encoded by SEQ ID NO: 227) 98+ 38.7 ### SEQ ID NO: 236 (encoded by SEQID NO: 235) 95 13.1 # SEQ ID NO: 238 (encoded by SEQ ID NO: 237) 98+22.3 ## SEQ ID NO: 240 (encoded by SEQ ID NO: 239) 95 not detected # SEQID NO: 270 (encoded by SEQ ID NO: 269) 40 4.6 # SEQ ID NO: 156 (encodedby SEQ ID NO: 155) 98+ 133.0 ###

Note that selectivities for R-MP of 98+% indicate that no S-MP wasdetected. Given the sensitivity of the CE assay, the results indicatethat the at least 98% of MP formed is the R-enantiomer. Thus, enzymesthat are listed as 98+% are at least 98% selective towards R-MP and maybe up to 100% selective.

Table 2 also shows the activity of the enzymes on a general aldolasesubstrate, CHA, as well as the relative expression of each enzyme, asdetermined by SDS-PAGE. Note that several enzymes did not showdetectable activity on CHA yet they did exhibit activity in making MP.

In summary, the aldolases show a wide range of activities, expressionand selectivities. Moreover, there are numerous aldolases that showexquisitely high selectivities (98% or greater) for R-MP.

Example 6 Discovery of Plant Pyruvate Aldolases

Degenerate PCR primers (see below) were designed and used to extractaldolase genes from cDNA prepared from Sclerochiton ilicifolius. The 5′and 3′ ends of the genes were recovered and the full length genes werethen PCR amplified.

SEQ ID NO: primer name Primer 335 F1 AARGTBTWYGARGACAATG (SEQ ID NO:335) 336 F2 GCDCAGAWCAAYGGRTGG (SEQ ID NO: 336) 337 R1CCATCRSYATCDGCRTADAGCCA (SEQ ID NO: 337) 338 R2 GCRTADAGCCAYTCNCCRTC(SEQ ID NO: 338)

Example 7 Cloning of Bacillus sphaericus D-Amino Acid Aminotransferase

The B. sphaericus D-amino acid aminotransferase (EC 2.6.1.21, also knownas D-alanine aminotransferase or D-aspartate aminotransferase) wasproduced recombinantly for use in coupled assays with the variousaldolases. This enzyme is homologous to D-aminotransferases describedpreviously for production of monatin (U.S. Publication No. 20040063175and U.S. Publication No. 20050282260).

Strains

B. sphaericus (ATCC number 10208) was grown on Nutrient Agar at 30° C.overnight. Groups of colonies were placed in 100 μL of sterile water andheated for 5 minutes at 95° C., to disrupt the cells. Three μL was usedin subsequent Polymerase Chain Reaction (PCR) amplifications.

Polymerase Chain Reaction Protocol

Primers were designed for cloning into pET 28b and pET 30a vectors(Novagen, Madison, Wis.), using the NcoI and BamHI sites. The pET 30construct contains an N-terminal His-tag and S-tag, whereas the pET 28construct is untagged.

Bacillus sphaericus dat primers: N term: (SEQ ID NO: 383)5′-GATATACCATGGCATACTCATTATGGAATG-3′ and C term: (SEQ ID NO: 384).5′-GTTATCGGATCCTTAGGCATTAATTGAAATTG-3′

The coding region was amplified using the following PCR protocol. In a50 μL reaction 3 μL template, 1.6 μM of each primer, 0.25 mM each dNTP,3.5 U Expand High Fidelity Polymerase (Roche, Indianapolis, Ind.), and1× Expand™ buffer with Mg were used. The thermocycler program usedincluded a hot start at 94° C. for 3 minutes, followed by 8 repetitionsof the following steps: 94° C. for 30 seconds, 52° C. for 30 seconds,and 72° C. for 2 minutes. Twenty-two subsequent cycles were done with anannealing temperature of 58° C. After 30 cycles, the sample wasmaintained at 72° C. for 7 minutes and then stored at 4° C. Clean PCRproducts of the correct size were obtained (approximately 850 bp for thedat gene).

Cloning

The PCR products were purified using the Qiagen QIAquick PCRpurification kit (Qiagen, Valencia, Calif.), and digested with BamHI andNcoI in BamHI buffer (New England Biolabs, Ipswich, Mass.).

Digested vector and inserts were purified using the Qiagen QIAquick GelExtraction Kit (Qiagen, Valencia, Calif.). Ligations were done using theRoche Rapid DNA Ligation Kit (Roche, Indianapolis, Ind.) and purifiedusing the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Theligations were transformed into Escherichia coli DH10B using a 0.2 cmcuvette and a Bio-Rad Gene Pulser II system as described in the Bio-Radelectroporation manual (Bio-Rad, Hercules, Calif.). The cells wereallowed to recover in 900 μL SOC medium for 30 minutes at 37° C. at 225rpm. Cells were plated on LB-agar plates containing kanamycin (25μg/mL).

Plasmid DNA was purified using the Qiagen spin miniprep kit (Qiagen,Valencia, Calif.) and screened for the correct inserts by restrictiondigest with BamHI and NcoI. The sequences of plasmids that appeared tohave the correct insert were verified by dideoxy chain termination DNAsequencing at Agencourt BioScience Corporation (Beverly, Mass.).Sequencing verified the coding sequence found in NCBI accession numberAF081278 Region: 134.985 (gi: 3513754), which produces a protein withamino acid sequence as listed in accession number AAC33964 (gi:3513755).

Gene Expression and Assays

Plasmid DNA was subcloned into E. coli expression host BL21(DE3)(Novagen, Madison, Wis.). The cultures were grown and the plasmids wereisolated using Qiagen miniprep kit (Qiagen, Valencia, Calif.), andanalyzed by restriction digest to confirm identity. Induction wastypically performed in LB medium containing kanamycin (50 μg/mL). Thecells were grown to an OD₆₀₀ of 0.4-0.8, induced with 0.1 mM IPTG(isopropyl thiogalacatoside) and sampled at 4 hours post induction. Cellextracts were prepared according to the protocol accompanying theNovagen BugBuster™ reagent (Novagen, Madison, Wis.) (with benzonasenuclease and Roche complete protease inhibitor cocktail added (Roche,Indianapolis, Ind.)). Very high levels of soluble protein were obtainedat the predicted molecular weight, as judged by SDS-PAGE. For somereactions, the pET 30 gene product was purified using His-Bindcartridges following manufacturer's protocols (Novagen, Madison, Wis.).The eluent fractions were desalted on PD-10 (GE Healthcare, Piscataway,N.J.) columns and eluted in 25-100 mM potassium phosphate buffer, pH7.5.

Cell extracts were analyzed for D-aminotransferase activity by followingproduction of alanine from pyruvate and D-tryptophan using the followingprotocol. One mL reactions were typically carried out in 100 mMpotassium phosphate buffer (pH 7.5), 50 μM pyridoxal phosphate, 25 mMsodium pyruvate, and 50 mM D-tryptophan. The reactions were initiated bythe addition of cell free extracts or purified enzyme and were incubated15 minutes-overnight at 30° C., with mild shaking. Formic acid was addedto a final concentration of two percent to stop the reaction, and theprecipitated protein was removed by centrifugation. Control reactionswithout added protein were also performed. Zero time points were alsoused as negative controls. Alanine was detected using OPA derivatizationas described in Example 1.

Example 8 Comparison of Total Monatin Production and IsomericDistribution for the Polypeptides with Aldolase Activity of SEQ ID NO:8,SEQ ID NO:4, SEQ ID NO:12, and SEQ ID NO:28, and C. testosteroni ProA

AT-103 transaminase (a broad specificity D-aminotransferase) waspurchased from BioCatalytics (Pasadena, Calif.) and either this enzymeor the recombinant enzyme produced in Example 7 was used in coupledreactions with HMG aldolases to produce monatin from D-tryptophan andpyruvate as described in U.S. Published Application No. 20050282260. TheProA aldolase from C. testosteroni was used as a benchmark aldolase forcomparative purposes, and was prepared as described in U.S. PublishedApplication No. 20040063175 and WO 03091396 A2. The aldolases testedwere isolated and transformed as described above in Example 4.

To produce test quantities of each aldolase, 50 mL cultures were grownin LB medium containing ampicillin (100 μg/mL), to an OD₆₀₀ ofapproximately 0.5. The strains containing SEQ ID NO:7, SEQ ID NO:3, andSEQ ID NO:11 constructs were induced with 100 μM of IPTG. The straincontaining the SEQ ID NO:27 construct was induced with 200 μg/Lanhydrotetracycline. The cells were grown 5 hours post-induction, andcellular extracts were prepared according to manufacturer's protocols(Novagen, Madison, Wis., Bugbuster reagent). Benzonuclease and proteaseinhibitor were also added. The soluble proteins in the cellular extractswere separated on a Bio-Rad Laboratories Experion AutomatedElectrophoresis Station (Bio-Rad, Hercules, Calif.) and analyzed forconcentration and percent expression using the Experion Software version1.1.98.0.

The following were added per 1 mL of reaction mixture: approximately 50μg aldolase (supplied in cellular extracts unless otherwise noted), 4 mMMgCl₂, 50 mM D-tryptophan, 0.5 mg purified B. sphaericusD-aminotransferase, 200 mM sodium pyruvate, 100 mM potassium phosphatebuffer pH 7.5, and 0.05 mM PLP. Experiments were run in duplicate, withnegative controls in which no aldolase was added. Samples were incubated1 hr, 2 hrs, and overnight (17-20 hours) at 30° C. with gentle shaking.Small amounts of monatin (<0.5 ppm) are produced without aldolase inovernight reactions, due to non-enzymatic reactions catalyzed bymagnesium and phosphate. Those values were subtracted from the numbersshown below, and averaged results are shown. The only stereoisomersdetected when producing monatin using these methods are R,R and S,R. Thepercent R,R is listed below, and was determined by reversed-phase LCpeak area.

TABLE 3 Total monatin produced from D-tryptophan and % R,R Aldolase(time point) Total monatin (ppm) % R,R monatin SEQ ID NO: 8 (1 hr) 15.6589.7 SEQ ID NO: 8 (18 hr) 129.22 79.0 SEQ ID NO: 4 (1 hr) 3.22 94.8 SEQID NO: 4 (18 hr) 12.14 93.8 SEQ ID NO: 12 (1 hr) 2.35 100 SEQ ID NO: 12(18 hr) 11.89 98.65 SEQ ID NO: 28 (1 hr) 14.70 100 SEQ ID NO: 28 (18 hr)95.14 97.35 C. testosteroni ProA (1 hr) 16.63 86.45 purified enzyme C.testosteroni ProA (18 hr) 86.86 63.1 purified enzyme

The SEQ ID NO:28 18 hour sample was also analyzed for stereoisomericdistribution by the FDAA derivatization method listed in Example 1,which yielded a result of 94.9% R,R and 5.1% S,R monatin.

The same experiments were done, side by side, using L-tryptophan as thestarting substrate and coupling the aldolases with HexAspC broadspecificity L-aminotransferase produced as described in U.S. PublishedApplication No. 20050282260 and purified. These reactions should yieldprimarily S,S monatin and R,S monatin. The reactions were alsosupplemented with 10 mM alpha-ketoglutarate as the amino acceptor forL-tryptophan transamination. Again, duplicate results are averaged belowfor total monatin (subtracting background levels without aldolasepresent), and percent S,S monatin is shown based on reversed phase LCpeak area. In some cases, because the aldolases are quite R-specific andproduce little total monatin, the reversed phase estimates ofstereoisomeric distribution are less accurate due to some tailing of thetryptophan peak that can co-elute with the S,S/R,R monatin peak. Thetrends are still informative in comparing R-specificity of thealdolases. Results from further analysis using the FDAA derivatizationmethod are shown in parentheses for several samples, and are moreaccurate. Total monatin numbers above approximately 400 ppm are higherthan the linear range of the scale of the standards used to quantitatethe results, so are qualitative results. The C. testosteroni ProAaldolase typically produces 95-100% S,S monatin, as shown in U.S.Published Application No. 20050282260.

TABLE 4 Total monatin produced from L-tryptophan and % S,S Aldolase(time point) Total monatin (ppm) % S,S monatin SEQ ID NO: 8 (1 hr)138.65 78.9 SEQ ID NO: 8 (18 hr) 600.3 78.15 SEQ ID NO: 4 (1 hr) belownegative control 95.65 SEQ ID NO: 4 (18 hr) 28.5 87.6 SEQ ID NO: 12 (1hr) below negative control 93.55 SEQ ID NO: 12 (18 hr) 24.9    75(59.35) SEQ ID NO: 28 (1 hr) 17.85 55.05 (18.9) SEQ ID NO: 28 (18 hr)135.5 27.25 (19.1) C. testosteroni ProA (1 hr) 440.35 92.5 purifiedenzyme C. testosteroni ProA (18 hr) 958.3 92.15 purified enzyme

One can see that the R-specificity of the polypeptide with aldolaseactivity of SEQ ID NO:28 is quite high compared to the benchmark ProAenzyme, this is also reflected in the low % S,S monatin produced,despite the high degree of specificity of the HexAspC aminotransferasefor S-MP in these reactions. The total monatin numbers, when comparingS,S monatin production versus R,R monatin production, are not indicativeof the aldolase activity. The D-aminotransferase is less active thanHexAspC, particularly at the concentrations of MP that are present inthese reactions.

For further comparison of the polypeptide with aldolase activity of SEQID NO:28 to the ProA enzyme from C. testosteroni, varying ratios ofD-aminotransferase to aldolase were utilized in reactions starting withD-tryptophan (no duplicate samples for these experiments). Reactionswere carried out as above. For reactions where aldolase concentrationwas kept constant, approximately 50 μg was used. For reactions whereD-aminotransferase was kept constant, 0.5 mg was used. For the 2 and 10mg/mL concentration of D-aminotransferase, lyophilized enzyme was used.For the 2 highest D-aminotransferase concentrations, duplicates wererun.

TABLE 5 Effect of D-aminotransferase concentration on R,R monatinproduction Concentration Total monatin of D-amino- (approximate % R,RAldolase transferase Time ppm) monatin SEQ ID NO: 28 0.25 mg/ml   1 hr 2100 SEQ ID NO: 28 0.25 mg/ml   overnight 141 97.1 SEQ ID NO: 28 0.5mg/ml   1 hr 8 100 SEQ ID NO: 28 0.5 mg/ml   overnight 273 96.5 SEQ IDNO: 28 1 mg/ml 1 hr 34 100 SEQ ID NO: 28 1 mg/ml overnight 638 96.5 SEQID NO: 28 2 mg/ml 1 hr 979 100 SEQ ID NO: 28 2 mg/ml overnight 1910 97.3SEQ ID NO: 28 10 mg/ml  1 hr 2930 99.1 SEQ ID NO: 28 10 mg/ml  overnight2950 96.5 C. testosteroni 0.25 mg/ml   1 hr 4 78.7 ProA (purified) C.testosteroni 0.25 mg/ml   overnight 257 61.1 ProA (purified) C.testosteroni 0.5 mg/ml   1 hr 25 79.0 ProA (purified) C. testosteroni0.5 mg/ml   overnight 480 62.5 ProA (purified) C. testosteroni 1 mg/ml 1hr 74 73.8 ProA (purified) C. testosteroni 1 mg/ml overnight 810 68.1ProA (purified) C. testosteroni 2 mg/ml 1 hr 325 73.1 ProA (purified) C.testosteroni 2 mg/ml overnight 2220 71.9 ProA (purified) C. testosteroni10 mg/ml  1 hr 2910 59.7 ProA (purified) C. testosteroni 10 mg/ml overnight 2450 67.5 ProA (purified) SEQ ID NO: 8 0.25 mg/ml   1 hr 492.3 SEQ ID NO: 8 0.25 mg/ml   overnight 219 69.8 SEQ ID NO: 8 0.5mg/ml   1 hr 14 84.9 SEQ ID NO: 8 0.5 mg/ml   overnight 426 67.5 SEQ IDNO: 8 1 mg/ml 1 hr 62 84.2 SEQ ID NO: 8 1 mg/ml overnight 877 68.7

For monatin levels above 400 ppm, the results are not in the linearrange of the standard curve and are approximate values only. The maximumamount of R,R monatin produced, when diluted appropriately, was 1100ppm. FDAA stereoisomeric analysis was done for the polypeptide withaldolase activity of SEQ ID NO:28 with 10 mg/mL D-aminotransferasesamples. At two hours, the sample contained 98.5% R,R monatin. At 17hours, the sample contained 95.9% R,R monatin. The polypeptide withaldolase activity of SEQ ID NO:28 produced high percentages of R,Rmonatin, even after long incubation times and using large amounts ofaminotransferase. If adequate D-aminotransferase is supplied, thepolypeptide with aldolase activity of SEQ ID NO:28 produces as muchtotal monatin as C. testosteroni ProA aldolase, indicating a similarspecific activity.

TABLE 6 Effect of aldolase concentration on R,R monatin productionConcentration of Total monatin % R,R Aldolase aldolase Time (ppm)monatin SEQ ID NO: 28 25 μg/ml 1 hr 7.0 100 SEQ ID NO: 28 25 μg/mlovernight 275 97.4 SEQ ID NO28 50 μg/ml 1 hr 9.0 97.3 SEQ ID NO: 28 50μg/ml overnight 334 95.7 SEQ ID NO: 28 100 μg/ml  overnight 297 93.3 C.testosteroni 25 μg/ml 1 hr 16 78.2 ProA (purified) C. testosteroni 25μg/ml overnight 491 73.2 ProA (purified) C. testosteroni 50 μg/ml 1 hr18 64.1 ProA (purified) C. testosteroni 50 μg/ml overnight 437 63.0 ProA(purified) C. testosteroni 100 μg/ml  1 hr 26 62.5 ProA (purified) C.testosteroni 100 μg/ml  overnight 513 61.5 ProA (purified) SEQ ID NO: 825 μg/ml 1 hr 11.0 88.1 SEQ ID NO: 8 25 μg/ml overnight 337.0 74.7 SEQID NO: 8 50 μg/ml 1 hr 14.0 78.2 SEQ ID NO: 8 50 μg/ml overnight 406.067.8 SEQ ID NO: 8 100 μg/ml  1 hr 24.0 70.1 SEQ ID NO: 8 100 μg/ml overnight 329.0 63.9

When the aldolase concentration is varied, there is not much of anincrease in total monatin. The percent R,R decreases with time and alsowith aldolase concentration, particularly when the D-aminotransferase islimiting.

To further examine the R-specificity of the aldolases tested,experiments were done starting with L-tryptophan and HexAspCaminotransferase, which was produced and purified as described in U.S.Published Application No. 20050282260. The HexAspC shows a strongselectivity for transamination of S-MP versus R-MP, thus percentagesabove 50% R,S monatin indicate a highly stereospecific aldolase. Ten mMalpha-ketoglutarate was supplied as an amino acceptor; however, at highconcentrations, pyruvate is also utilized by the L-aminotransferase. Inthese reactions, typically only S,S and R,S monatin are produced withinthe limits of detection of the FDAA derivatization protocol.

TABLE 7 Effect of L-aminotransferase concentration on S,S monatinproduction Concentration Total monatin of D-amino- (approximate % S,SAldolase transferase Time ppm) monatin SEQ ID NO: 28 0.25 mg/ml 1 hr 1333.8 SEQ ID NO: 28 0.25 mg/ml overnight 127 34.2 SEQ ID NO: 28  0.5mg/ml 1 hr 31 30.9 SEQ ID NO: 28  0.5 mg/ml overnight 272 26.8 SEQ IDNO: 28   1 mg/ml 1 hr 34 20.3 SEQ ID NO: 28   1 mg/ml overnight 385 23.5C. testosteroni 0.25 mg/ml 1 hr 523 94.2 ProA (purified) C. testosteroni0.25 mg/ml overnight 1817 93.7 ProA (purified) C. testosteroni  0.5mg/ml 1 hr 602 91.8 ProA (purified) C. testosteroni  0.5 mg/ml overnight2122 89.9 ProA (purified) C. testosteroni   1 mg/ml 1 hr 873 90.2 ProA(purified) C. testosteroni   1 mg/ml overnight 1237 82.6 ProA (purified)SEQ ID NO: 8 0.25 mg/ml 1 hr 339 86.3 SEQ ID NO: 8 0.25 mg/ml overnight1499 88.0 SEQ ID NO: 8  0.5 mg/ml 1 hr 211 80.3 SEQ ID NO: 8  0.5 mg/mlovernight 1328 83.1 SEQ ID NO: 8   1 mg/ml 1 hr 400 74.6 SEQ ID NO: 8  1 mg/ml overnight 1370 79.0

TABLE 8 Effect of aldolase concentration on S,S monatin productionConcentration Total monatin % S,S Aldolase of aldolase Time (ppm)monatin SEQ ID NO: 28 25 μg/ml 1 hr 11 25.1 SEQ ID NO: 28 25 μg/mlovernight 112 20.0 SEQ ID NO: 28 50 μg/ml 1 hr 18 31.8 SEQ ID NO: 28 50μg/ml overnight 160 27.0 SEQ ID NO: 28 100 μg/ml  1 hr 33 33.2 SEQ IDNO: 28 100 μg/ml  overnight 238 41.4 C. testosteroni 25 μg/ml 1 hr 30586.4 ProA (purified) C. testosteroni 25 μg/ml overnight 1094 87.5 ProA(purified) C. testosteroni 50 μg/ml 1 hr 575 90.9 ProA (purified) C.testosteroni 50 μg/ml overnight 1449 89.5 ProA (purified) C.testosteroni 100 μg/ml  1 hr 817 93.6 ProA (purified) C. testosteroni100 μg/ml  overnight 1360 89.7 ProA (purified) SEQ ID NO: 8 25 μg/ml 1hr 134 70.7 SEQ ID NO: 8 25 μg/ml overnight 728 76.3 SEQ ID NO: 8 50μg/ml 1 hr 197 80.0 SEQ ID NO: 8 50 μg/ml overnight 928 81.4 SEQ ID NO:8 100 μg/ml  1 hr 279 86.7 SEQ ID NO: 8 100 μg/ml  overnight 1383 86.8

For aldolases that are highly R-specific, such as the polypeptide withaldolase activity of SEQ ID NO:28, less total monatin is produced andincreasing the amount of aldolase does increase total monatin (as wellas % S,S). These aldolases produce less S-MP substrate, the preferredsubstrate for the L-aminotransferase used. For enzymes that are lessR-specific, such as ProA, increasing aldolase does not significantlyimprove total monatin production or % S,S monatin. Increasing the amountof L-aminotransferase added decreases the percentage of S,S monatinproduced. Based on the above analysis, the polypeptide with aldolaseactivity of SEQ ID NO:8 is between ProA and the polypeptide withaldolase activity of SEQ ID NO:28 in terms of R-specificity, whichagrees with data above where % R-MP is measured for the aldol stepalone.

Subcloning of SEQ ID NO:27

The following primers were used to PCR amplify the aldolase gene:5′-gaggagctcgagtcagacgtatttcagtcctttttc-3′ (SEQ ID NO:385) and5′-agaagacatatgatttatcagccggggac-3′ (SEQ ID NO:386). The aldolase geneSEQ ID NO:27 encodes the polypeptide with aldolase activity of SEQ IDNO:28. The resulting PCR product was digested with XhoI and NdeI to cutat the sites that had been engineered into the primers. The fragment wasgel purified (QIAquick Gel extraction Kit (Qiagen, Valencia, Calif.))and ligated (using T4 DNA ligase) with pET28b that had been digestedwith XhoI and NdeI and gel purified. The ligation was transformed intoTOP10F′ chemically competent cells. Colonies growing on the plates werescreened for inserts and several isolates with inserts were submittedfor DNA sequence analysis (Agencourt, Beverly, Mass.).

Purification of the Polypeptide with Aldolase Activity of SEQ ID NO:28

Confirmed aldolase clones were transformed into either BL21 DE3 or BL21DE3 pLysS. Overnight cultures grown with the appropriate antibiotic werediluted into fresh media (typically 1:100) and grown to an OD₆₀₀˜0.6with aeration at 37° C. Cultures were then induced with 1 mM IPTG andshifted to 30° C. (with aeration) and incubation was continuedovernight. Cells were harvested by centrifugation. The cell pellet wastypically subjected to one freeze thaw cycle to assist with cell lysis.The cell pellet was lysed in BugBuster and Benzonase (Novagen, Madison,Wis.) (according to the manufacturer's protocol). Cell debris wasremoved by centrifugation. The crude protein extract was applied to aHisBind column (Novagen, Madison, Wis.) that had been prepared accordingto the manufacturer's protocol. The column was washed and protein waseluted according to the manufacturer's protocol. The purified proteinwas desalted with PD-10 columns (GE Healthcare, Piscataway, N.J.). Thebuffer used for the exchange was 50 mM potassium phosphate pH 7.5, 100mM NaCl, 4 mM MgCl₂. Purified protein was concentrated with Amiconcentrifugal concentrators (Millipore, Billerica, Mass.).

Example 9 Comparison of Total Monatin Production and IsomericDistribution for Polypeptides with Aldolase Activity of SEQ ID NO:40,SEQ ID NO:298, SEQ ID NO:36, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:96,SEQ ID NO:54, SEQ ID NO:122, SEQ ID NO:142, SEQ ID NO:42, SEQ ID NO:130,SEQ ID NO:112, SEQ ID NO:108, SEQ ID NO:94, SEQ ID NO:80, and SEQ IDNO:28

AT-103 transaminase (a broad specificity D-aminotransferase) waspurchased from BioCatalytics (Pasadena, Calif.) and either this enzymeor the recombinant enzyme produced in Example 7 was used in coupledreactions with HMG aldolases to produce monatin from D-tryptophan andpyruvate as described in U.S. Published Application No. 20050282260. Thepolypeptide with aldolase activity of SEQ ID NO:28 (his-tagged) was usedas a benchmark aldolase for comparative purposes, and was produced andpurified as described at the end of Example 8. The other aldolasestested were isolated and transformed as described above in Example 4.

To produce test quantities of each aldolase, 25 mL cultures were grownin LB medium containing ampicillin (100 μg/mL), to an OD₆₀₀ ofapproximately 0.4. The strains were induced with 100 μM of IPTG. Thecells were grown 4 hours post-induction, and cellular extracts wereprepared according to manufacturer's protocols (Novagen, Madison, Wis.,Bugbuster reagent) with benzonuclease. The soluble proteins in thecellular extracts were separated on a Bio-Rad Laboratories ExperionAutomated Electrophoresis Station (Bio-Rad, Hercules, Calif.) andanalyzed for concentration and percent expression using the ExperionSoftware version 1.1.98.0.

The following were added per 1 mL of reaction mixture: approximately 50μg aldolase (supplied in cellular extracts unless otherwise noted), 4 mMMgCl₂, 50 mg/mL D-tryptophan, 2 mg AT-103 (BioCatalytics, Pasadena,Calif.), 200 mM sodium pyruvate, 100 mM potassium phosphate buffer pH7.5, and 0.05 mM PLP. The D-tryptophan is not soluble at this higherconcentration, but was used to ensure that the reactions were kept atsaturating amounts of D-tryptophan. Experiments were run in duplicate,with negative controls in which no aldolase was added. Samples wereincubated 2 hrs and overnight (17-20 hours) at 30° C. with gentleshaking. Small amounts of monatin are produced overnight withoutaldolase (˜0.5 ppm), due to non-enzymatic reactions catalyzed bymagnesium and phosphate. Typical values for % R,R monatin are 50% forthese samples. The negative control values were subtracted from thenumbers shown below, and averaged results are shown. The onlystereoisomers detected when producing monatin using these methods areR,R and S,R. The percent R,R is listed below, and was determined byreversed-phase LC peak area. The same experiment was conducted afterstorage of the cell extracts and the purified polypeptide with aldolaseactivity of SEQ ID NO:28 for 2 months at −20° C., this time 50 mMD-tryptophan was used as in Example 8. Twice the amount of aldolase wasadded with the exception of the polypeptide with aldolase activity ofSEQ ID NO:28, for which approximately 50 μg was utilized again. Theseresults are shown to the right of Table 9. FDAA derivatization resultsfor isomeric distribution are shown in parentheses.

TABLE 9 Total monatin produced from D-tryptophan and % R,R Total Totalmonatin % R,R monatin % R,R Aldolase (time point) (ppm) monatin (ppm)monatin SEQ ID NO: 40 (2 hr) 336 82.7 238 66.8 SEQ ID NO: 40. (18 hr)707.55 76.2 748.5 62.4 SEQ ID NO: 298 (2 hr) 394.3 98.0 183 91.9 (91.6)SEQ ID NO: 298 (18 hr) 819.5 96.1 648.5 80.0 SEQ ID NO: 36 (2 hr) 5698.2 52.5 94.0 SEQ ID NO: 36 (18 hr) 123.25 96.9 296 88.5 SEQ ID NO: 62(2 hr) 1.15 78.4 0 n/a SEQ ID NO: 62 (18 hr) 0.95 73.8 16 89.2 SEQ IDNO: 64 (2 hr) 16.7 98.8 24 96.9 SEQ ID NO: 64 (18 hr) 43.7 97.6 161 98.5SEQ ID NO: 96 (2 hr) 30.4 99.2 29 96.0 SEQ ID NO: 96 (18 hr) 80.8 98.3200 97.3 SEQ ID NO: 54 (2 hr) 183.1 99.4 135.5 98.2 (98.8) SEQ ID NO: 54(18 hr) 457.7 98.9 488.5 96.9 SEQ ID NO: 122 (2 hr) 129.3 97.9 126 97.8(99.1) SEQ ID NO: 122 (18 hr) 289.85 95.8 471.5 94.4 SEQ ID NO: 142 (2hr) 40.4 98.3 58.5 95.9 SEQ ID NO: 142 (18 hr) 82.3 97.3 388 96.8 SEQ IDNO: 42 (2 hr) 335.9 98.2 206.5 93.3 SEQ ID NO: 42 (18 hr) 612.45 96.6630.5 82.9 SEQ ID NO: 130 (2 hr) 77.5 99.3 60.5 98.5 (99.6) SEQ ID NO:130 (18 hr) 177.45 99.1 368.5 98.4 SEQ ID NO: 112 (2 hr) 20.4 99.0 2798.6 SEQ ID NO: 112 (18 hr) 57.75 98.3 186.5 99.3 SEQ ID NO: 108 (2 hr)44.4 98.7 41 97.0 SEQ ID NO: 108 (18 hr) 111.7 98.0 265.5 93.3 (96.4)SEQ ID NO: 94 (2 hr) 69.4 98.2 56 94.4 SEQ ID NO: 94 (18 hr) 181.95 96.9341 84.8 SEQ ID NO: 80 (2 hr) 27.9 98.9 29.5 98.8 SEQ ID NO: 80 (18 hr)74 97.9 219 96.6 SEQ ID NO: 28 - purified 131.3 99.5 53 96.7 (99.6) (2hr) SEQ ID NO: 28 - purified 407.4 99.2 257 99.2 (18 hr)

One can see that certain enzymes are more stable to storage than otheraldolases, based on ratios of activity. A secondary product, most likely4-hydroxy-4-methyl glutamate can also be formed during these reactions.The enzymes above were ranked for their specificity towards monatinproduction by comparing the peak areas of that versus the byproduct. Theresults were the polypeptide with aldolase activity of SEQ ID NO:122>SEQID NO:42>SEQ ID NO:80>SEQ ID NO:108>SEQ ID NO:96>SEQ ID NO:112>SEQ IDNO:130>SEQ ID NO:36>SEQ ID NO:94>SEQ ID NO:298>SEQ ID NO:40>SEQ IDNO:142>SEQ ID NO:54>SEQ ID NO:64>SEQ ID NO:28>SEQ ID NO:62.

Based on initial experiments, the polypeptides with aldolase activity ofSEQ ID NO:298, SEQ ID NO:54, and SEQ ID NO:42 looked the most promisingin terms of activity level and % R,R monatin produced. These enzymeswere subcloned into pET expression vectors with and without his-tags.

Cloning of SEQ ID NO:297, SEQ ID NO:53, and SEQ ID NO:41.

Primers used for cloning:

TABLE 10 SEQ ID NO: 5′ primer 3′primer 297 5′-agaagacatatgggtgtcgt5′-ataataggatccttagac cgtccaaaac-3′ atatttgaggccc-3′ (SEQ ID NO: 387)(SEQ ID NO: 388) 53 5′-ataatacatatgaagcc 5′-agaagaggatccttagaggtggtggtgc-3′ cataggtgagcccc-3′ (SEQ ID NO: 389) (SEQ ID NO: 390) 415′-ataataccatgggtgtc 5′-agaagaggatccttaga gtggtccag-3′catatttcaggcccc-3′ (SEQ ID NO: 391) (SEQ ID NO: 392)

SEQ ID NO:297, SEQ ID NO:53, and SEQ ID NO:41 were amplified by PCR anddigested with appropriate enzymes (NdeI and BamHI for the PCR productscontaining SEQ ID NO:297 and SEQ ID NO:53, NcoI and BamHI for the PCRproducts containing SEQ ID NO:41) and gel purified (QIAquick Gelextraction Kit (Qiagen, Valencia, Calif.)). SEQ ID NO:297 and SEQ IDNO:53 were individually ligated into pET28 that had been digested withNdeI and BamHI and gel purified. SEQ ID NO:41 was ligated to pET30 thathad been digested with NcoI and BamHI and gel purified. The ligation wastransformed into TOP10. Colonies were screened for inserts. Isolateswith an insert were submitted for DNA sequence analysis (Agencourt,Beverly, Mass.).

Purification of Aldolases

Confirmed aldolase clones were transformed into either BL21 DE3 or BL21DE3 pLysS. Overnight cultures grown with the appropriate antibiotic werediluted into fresh media (typically 1:100) and grown to an OD₆₀₀˜0.6with aeration at 37° C. Cultures were then induced with 1 mM IPTG andshifted to 30° C. (with aeration) and incubation was continuedovernight. Cells were harvested by centrifugation. The cell pellet wastypically subjected to one freeze thaw cycle to assist with cell lysis.The cell pellet was lysed in BugBuster and Benzonase (Novagen, Madison,Wis.) (according to the manufacturer's protocol). Cell debris wasremoved by centrifugation. The crude protein extract was applied to aHisBind column (Novagen, Madison, Wis.) that had been prepared accordingto the manufacturer's protocol. The column was washed and protein waseluted according to the manufacturer's protocol. The purified proteinwas desalted with PD-10 columns (GE Healthcare, Piscataway, N.J.). Thebuffer used for the exchange was 50 mM potassium phosphate pH 7.5, 100mM NaCl, 4 mM MgCl₂. Purified protein was concentrated with Amiconcentrifugal concentrators (Millipore, Billerica, Mass.).

Testing of Purified Aldolases

Purified aldolases were tested for their ability to produce R,R monatinfrom D-tryptophan. The following were added per 1 mL of reactionmixture: approximately 50 μg purified aldolase, 4 mM MgCl₂, 50 mMD-tryptophan, 0.5 mg purified B. sphaericus D-aminotransferase, 200 mMsodium pyruvate, 100 mM potassium phosphate buffer pH 7.5, and 0.05 mMPLP. Samples were taken at 2 hours and overnight. Results are shown inTable 11 below.

TABLE 11 Total monatin produced from D-tryptophan and % R,R Total % R,Rmonatin % R,R monatin monatin (Reversed Phase (FDAA Aldolase (timepoint) (ppm) LC peak area) derivatization) SEQ ID NO: 298 (2 hr) 16.9588.5 n/a SEQ ID NO: 298 212 77.6 71   (overnight) SEQ ID NO: 54 (2 hr)12.05 96.7 n/a SEQ ID NO: 54 (overnight) 161.85 93.0 91.1 SEQ ID NO: 42(2 hr) 20.95 80.3 n/a SEQ ID NO: 42 (overnight) 223.2 69.1 62.1 SEQ IDNO: 28 (2 hr) 14.25 95.8 n/a SEQ ID NO: 28 (overnight) 176.6 93.4 92.3

The same experiments were done using L-tryptophan as the startingsubstrate and coupling the aldolases with HexAspC broad specificityL-aminotransferase produced and purified as described in U.S. PublishedApplication No. 20050282260 (0.5 mg of purified protein). Results areshown below in Table 12 for total monatin production (subtractingbackground levels without aldolase present), and percent S,S monatin isshown based on reversed phase LC peak area. Numbers above 400 ppm areoutside the linear range of the standard curve, and are approximate.

TABLE 12 Total monatin produced from L-tryptophan and % S,S Total % S,Smonatin % S,S monatin monatin (Reversed Phase (FDAA Aldolase (timepoint) (ppm) LC peak area) derivatization) SEQ ID NO: 298 (1 hr) 186.664.0 n/a SEQ ID NO: 298 197.5 64.3 67.6 (overnight) SEQ ID NO: 54 (1 hr)70.4 36.5 n/a SEQ ID NO: 54 (overnight) 87.8 41.7 42.1 SEQ ID NO: 42 (1hr) 401.1 80.9 n/a SEQ ID NO: 42 (overnight) 507.5 82.9 85.8 SEQ ID NO:28 (1 hr) 56.2 30.1 n/a SEQ ID NO: 28 (overnight) 88.8 32.2 33.8

These data and the above R,R monatin data illustrate that for R-MPspecificity, the polypeptides with aldolase activity have the followingorder: SEQ ID NO:28>SEQ ID NO:54>SEQ ID NO:298>SEQ ID NO:42.

Example 10 Comparison of Total Monatin Production and IsomericDistribution for the Polypeptides with Aldolase Activity of SEQ IDNO:116, SEQ ID NO:76, SEQ ID NO:44, SEQ ID NO:148, SEQ ID NO:46, SEQ IDNO:134, SEQ ID NO:74, SEQ ID NO:126, SEQ ID NO:102, SEQ ID NO:58, SEQ IDNO:88, SEQ ID NO:50, SEQ ID NO:106, SEQ ID NO:304, SEQ ID NO:300, andSEQ ID NO:28

The recombinant enzyme produced in Example 7 was used in coupledreactions with HMG aldolases to produce monatin from D-tryptophan andpyruvate as described in U.S. Published Application No. 20050282260. Thepolypeptide with aldolase activity of SEQ ID NO:28 was used as abenchmark in these assays and had been purified as described in Example8.

To produce test quantities of each aldolase, 25 mL cultures were grownin LB medium containing ampicillin (100 μg/mL), to an OD₆₀₀ ofapproximately 0.5. The cultures were induced with 1 mM of IPTG. Thecells were shifted to 30° C. and were grown overnight. Cellular extractswere prepared according to manufacturer's protocols (Novagen, Madison,Wis., Bugbuster reagent). Benzonuclease was also added. The solubleproteins in the cellular extracts were separated on a Bio-RadLaboratories Experion Automated Electrophoresis Station (Bio-Rad,Hercules, Calif.) and analyzed for concentration and percent expressionusing the Experion Software version 1.1.98.0.

The following were added per 1 mL of reaction mixture: approximately 50μg aldolase (supplied in cellular extracts unless otherwise noted), 4 mMMgCl₂, 50 mM D-tryptophan, 0.5 mg purified B. sphaericusD-aminotransferase, 200 mM sodium pyruvate, 100 mM potassium phosphatebuffer pH 7.5, and 0.05 mM PLP. Dithiothreitol (“DTT”) was added (finalconcentration 2 mM) to the samples noted below. Experiments were run induplicate. Samples were incubated 2 hrs, and overnight (20 hours) at 30°C. with gentle shaking. Averaged results are shown below. The onlystereoisomers detected when producing monatin using these methods areR,R and S,R. The percent R,R is listed below, and was determined byreversed-phase LC peak area.

TABLE 13 Total monatin produced from D-tryptophan and % R,R Aldolase(time point) Total monatin (ppm) % R,R monatin SEQ ID NO: 116 (2 hr)34.5 97 SEQ ID NO: 116 (18 hr) 99 95 SEQ ID NO: 76 (2 hr) 40 76 SEQ IDNO: 76 (18 hr) 112 67 SEQ ID NO: 44 (2 hr) 32.5 97 SEQ ID NO: 44 (18 hr)93.5 94 SEQ ID NO: 148 (2 hr) 31.5 94 SEQ ID NO: 148 (18 hr) 98 89 SEQID NO: 46 (2 hr) 42.5 84 SEQ ID NO: 46 (18 hr) 169 72 SEQ ID NO: 134 (2hr) 43.5 92 SEQ ID NO: 134 (18 hr) 113 86 SEQ ID NO: 74 (2 hr) 23.5 96SEQ ID NO: 74 (18 hr) 78.5 92 SEQ ID NO: 126 (2 hr) 18 94 SEQ ID NO: 126(18 hr) 72 92 SEQ ID NO: 102 (2 hr) 1 0 SEQ ID NO: 102 (18 hr) 4.5 91SEQ ID NO: 58 (2 hr) 23 92 SEQ ID NO: 58 (18 hr) 122 88 SEQ ID NO: 88 (2hr) 57.5 74 SEQ ID NO: 88 (18 hr) 200.5 64 SEQ ID NO: 50 (2 hr) 32.5 99SEQ ID NO: 50 (18 hr) 131.5 97 SEQ ID NO: 106 (2 hr) 4.5 78 SEQ ID NO:106 (18 hr) 20 95 SEQ ID NO: 304 (2 hr) 0 0 SEQ ID NO: 304 (18 hr) 0.4555 SEQ ID NO: 304 DTT (2 hr) 0 0 SEQ ID NO: 304 DTT (18 hr) 0.55 53 SEQID NO: 300 (2 hr) 0.85 34 SEQ ID NO: 300 (18 hr) 5.5 36 SEQ ID NO: 300DTT (2 hr) 1.5 55 SEQ ID NO: 300 DTT (18 hr) 9 40 SEQ ID NO: 28 (2 hr)25 99 SEQ ID NO: 28 (18 hr) 69 97

Total monatin production numbers ranged from 1 ppm to over 200 ppm and %R,R ranged from 0% to 99%. Because the aminotransferase was the same forall of the aldolases, changing the aldolase can have a significantimpact on both the amount of monatin produced and the stereoisomericdistribution of the monatin produced. DTT (as in the samples below)appeared to increase the amount of total monatin produced.

The same experiments as above were done using L-tryptophan as thestarting substrate and coupling the aldolases (supplied as cellularextract) with HexAspC broad specificity L-aminotransferase partiallypurified (0.5 mg of HexAspC). Averaged results (of duplicates) are shownbelow in Table 14 for total monatin production (subtracting backgroundlevels without aldolase present), and percent S,S monatin is shown basedon reversed phase LC peak area. Numbers above 400 ppm are outside thelinear range of the standard curve, and are approximate. Purifiedpolypeptide with aldolase activity of SEQ ID NO:28 was used as abenchmark. Polypeptides with aldolase activity of SEQ ID NO:304 and SEQID NO:300 (plant derived) were used with and without 2 mM DTT. Shannonand Marcus (The Journal of Biological Chemistry 237: 3342-3347, 1962)used mercaptoethanol as a reducing agent in the original purification ofa peanut HMG aldolase.

TABLE 14 Total monatin produced from L-tryptophan and % S,S Aldolase(time point) Total monatin (ppm) % S,S monatin SEQ ID NO: 116 (2 hr) 12947.9 SEQ ID NO: 116 (21 hr) 207 56.4 SEQ ID NO: 76 (2 hr) 949 90.6 SEQID NO: 76 (21 hr) 1181 89.0 SEQ ID NO: 44 (2 hr) 128 55.0 SEQ ID NO: 44(21 hr) 237 61.7 SEQ ID NO: 148 (2 hr) 199 71.5 SEQ ID NO: 148 (21 hr)358 74.4 SEQ ID NO: 46 (2 hr) 346 79.3 SEQ ID NO: 46 (21 hr) 757 83.3SEQ ID NO: 134 (2 hr) 215 69.2 SEQ ID NO: 134 (21 hr) 370 74.1 SEQ IDNO: 74 (2 hr) 75 51.4 SEQ ID NO: 74 (21 hr) 137 58.8 SEQ ID NO: 126 (2hr) 47 56.7 SEQ ID NO: 126 (21 hr) 113 56.7 SEQ ID NO: 102 (2 hr) sameas control n/a SEQ ID NO: 102 (21 hr) 11.5 61.1 SEQ ID NO: 58 (2 hr) 11371.9 SEQ ID NO: 58 (21 hr) 351 75.5 SEQ ID NO: 88 (2 hr) 852 90.1 SEQ IDNO: 88 (21 hr) 1352 88.8 SEQ ID NO: 50 (2 hr) 62 30.8 SEQ ID NO: 50 (21hr) 145 38.6 SEQ ID NO: 106 (2 hr) 3.5 31.0 SEQ ID NO: 106 (21 hr) 4534.4 SEQ ID NO: 304 (2 hr) same as control n/a SEQ ID NO: 304 + DTT (2hr) 1 n/a SEQ ID NO: 304 (21 hr) same as control n/a SEQ ID NO: 304 +DTT (21 hr) 10 n/a SEQ ID NO: 300 (2 hr) 73 75.2 SEQ ID NO: 300 + DTT (2hr) 121 83   SEQ ID NO: 300 (21 hr) 91 63.6 SEQ ID NO: 300 + DTT (21 hr)197 71.6 SEQ ID NO: 28 (2 hr) 55 35.1 SEQ ID NO: 28 (21 hr) 87 40.4

Example 11 Effect of Dithiothreitol (DTT) on Monatin Production

Several of the enzymes in Example 10 were chosen for further study. Theplant derived aldolases showed improvement upon the addition of DTT as areducing agent. It was noted that the microbially-derived aldolases fromenvironmental samples also contain a high percentage of cysteineresidues. Therefore, further experiments were conducted to see if DTTincreased monatin production for non-plant aldolases as well.

The following were added per 1 mL of reaction mixture: approximately 50μg aldolase (supplied in cellular extracts unless otherwise noted), 4 mMMgCl₂, 50 mM D-tryptophan, 2 mg AT-103, 200 mM sodium pyruvate, 100 mMpotassium phosphate buffer pH 7.5, and 0.05 mM PLP. Dithiothreitol wasadded (final concentration 2 mM) to the samples noted below. Experimentswere run in duplicate. Samples were incubated 2 hrs at 30° C. withgentle shaking. Averaged results are shown below for total monatin asdetermined by LC/MS/MS, with the background production of monatin (noaldolase control) subtracted.

TABLE 15 Total monatin produced from D-tryptophan Aldolase Total monatin(ppm) SEQ ID NO: 116 126 SEQ ID NO: 116 + DTT 102 SEQ ID NO: 44 107 SEQID NO: 44 + DTT 103 SEQ ID NO: 46 88 SEQ ID NO: 46 + DTT 161 SEQ ID NO:58 118 SEQ ID NO: 58 + DTT 141 SEQ ID NO: 50 243 SEQ ID NO: 50 + DTT 170SEQ ID NO: 28 purified protein 174 SEQ ID NO: 28 + DTT purified protein196

The no aldolase control produced 10 ppm of total monatin with andwithout DTT, indicating that the DTT is not affecting the overallreaction by reduction of byproducts, and is not affecting theD-aminotransferase activity. The polypeptides with aldolase activity ofSEQ ID NO:46, SEQ ID NO:58, and SEQ ID NO:28 all showed a benefit fromthe addition of DTT. The polypeptide with aldolase activity of SEQ IDNO:46 showed the highest benefit, approximately 1.8 fold higher activitywith 2 mM DTT. Two polypeptides with aldolase activity appear to havebeen inhibited by DTT (SEQ ID NO:116 and SEQ ID NO:50) while no effectwas noted, within experimental error, for the polypeptide with aldolaseactivity of SEQ ID NO:44. However, it is possible that for each aldolaseutilized there is an optimal concentration of DTT in order to detect abenefit of providing the reducing agent.

The polypeptide with aldolase activity of SEQ ID NO:88 was chosen tostudy the effect of DTT concentration on monatin production. Coupledreactions were carried out as above. Results are plotted in FIG. 15. Theoptimal concentration of DTT in this assay was between 2.5 and 5 mM, forthe amount of aldolase added. Interestingly, if no DTT was added theamount of monatin produced was almost as high as the 2.5 mM DTT, butadding suboptimal amounts of DTT (0.5-1 mM) actually appears to beinhibitory, as well as addition of too much DTT (20 mM).

Example 12 Comparison of Total Monatin Production and IsomericDistribution for the Polypeptides with Aldolase Activity of SEQ IDNO:278, SEQ ID NO:162, SEQ ID NO:276, SEQ ID NO:178, SEQ ID NO:202, SEQID NO:166, SEQ ID NO:218, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:244,SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:264, SEQ ID NO:268, SEQ IDNO:272, SEQ ID NO:184, SEQ ID NO:282, SEQ ID NO:186, SEQ ID NO:192, SEQID NO:200, SEQ ID NO:280, SEQ ID NO:284, SEQ ID NO:172, SEQ ID NO:180,SEQ ID NO:168, SEQ ID NO:228, SEQ ID NO:236, SEQ ID NO:238, SEQ IDNO:240, SEQ ID NO:270, SEQ ID NO:156, and SEQ ID NO:28

The recombinant enzyme produced in Example 7 was used in coupledreactions with HMG aldolases to produce monatin from D-tryptophan andpyruvate as described in U.S. Published Application No. 20050282260. Thepolypeptide with aldolase activity of SEQ ID NO:28 was used as abenchmark in these assays and had been purified as described in Example8.

To produce test quantities of each aldolase, 25 mL cultures were grownin LB medium containing ampicillin (100 μg/mL), to an OD₆₀₀ ofapproximately 0.5. The cultures were induced with 1 mM of IPTG. Thecells were shifted to 30° C. and were grown overnight. Cellular extractswere prepared using Bugbuster reagent according to manufacturer'sprotocols (Novagen, Madison, Wis.). Benzonuclease was also added. Thesoluble proteins in the cellular extracts were separated on a Bio-RadLaboratories Experion Automated Electrophoresis Station (Bio-Rad,Hercules, Calif.) and analyzed for concentration and percent expressionusing the Experion Software version 1.1.98.0.

The following were added per 1 mL of reaction mixture: approximately 200μg of the polypeptides with aldolase activity of SEQ ID NO:278, SEQ IDNO:162, SEQ ID NO:276, SEQ ID NO:178, SEQ ID NO:202, SEQ ID NO:166, SEQID NO:218, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:244, SEQ ID NO:250,SEQ ID NO:252, SEQ ID NO:264, SEQ ID NO:268, SEQ ID NO:272, SEQ IDNO:184, SEQ ID NO:282, SEQ ID NO:186, SEQ ID NO:192, and SEQ ID NO:200or 50 μg of the polypeptide with aldolase activity of SEQ ID NO:280, SEQID NO:284, SEQ ID NO:172, SEQ ID NO:180, SEQ ID NO:168, SEQ ID NO:228,SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:270, and SEQ IDNO:156 (supplied in cellular extracts unless otherwise noted), 4 mMMgCl₂, 50 mM D-tryptophan, 0.5 mg purified B. sphaericusD-aminotransferase, 200 mM sodium pyruvate, 100 mM potassium phosphatebuffer pH 7.5, and 0.05 mM PLP. Experiments were run in duplicate.Samples were incubated 2 hrs, and overnight (20 hours) at 30° C. withgentle shaking. Averaged results are shown below. The only stereoisomersdetected when producing monatin using these methods are R,R and S,R. Thepercent R,R is listed below, and was determined by reversed-phase LCpeak area.

TABLE 16 Total monatin produced from D-tryptophan and % R,R Aldolase(time point) Total monatin (ppm) % R,R monatin SEQ ID NO: 278 (1 hr)11.35 100 SEQ ID NO: 278 (18 hr) 282.15 96 SEQ ID NO: 162 (1 hr) 19.35100 SEQ ID NO: 162 (18 hr) 277.9 98 SEQ ID NO: 276 (1 hr) 27.2 100 SEQID NO: 276 (18 hr) 421 98 SEQ ID NO: 178 (1 hr) 24.8 98 SEQ ID NO: 178(18 hr) 394.25 94 SEQ ID NO: 202 (1 hr) 0 0 SEQ ID NO: 202 (18 hr) 19.291 SEQ ID NO: 166 (1 hr) 42.8 89 SEQ ID NO: 166 (18 hr) 601.25 71 SEQ IDNO: 218 (1 hr) 15.6 99 SEQ ID NO: 218 (18 hr) 456.05 96 SEQ ID NO: 224(1 hr) 19.7 98 SEQ ID NO: 224 (18 hr) 406.55 93 SEQ ID NO: 226 (1 hr)41.3 95 SEQ ID NO: 226 (18 hr) 460.15 84 SEQ ID NO: 244 (1 hr) 11.6 99SEQ ID NO: 244 (18 hr) 168.3 98 SEQ ID NO: 250 (1 hr) 20.25 95 SEQ IDNO: 250 (18 hr) 289.25 89 SEQ ID NO: 252 (1 hr) 48.4 81 SEQ ID NO: 252(18 hr) 335.8 73 SEQ ID NO: 264 (1 hr) 31.65 82 SEQ ID NO: 264 (18 hr)252.35 77 SEQ ID NO: 268 (1 hr) 12.95 98 SEQ ID NO: 268 (18 hr) 252.5595 SEQ ID NO: 272 (1 hr) 13.8 98 SEQ ID NO: 272 (18 hr) 165.8 98 SEQ IDNO: 184 (1 hr) 19.55 96 SEQ ID NO: 184 (18 hr) 221.85 94 SEQ ID NO: 282(1 hr) 29.75 95 SEQ ID NO: 282 (18 hr) 399.05 91 SEQ ID NO: 186 (1 hr)14.4 94 SEQ ID NO: 186 (18 hr) 116.15 93 SEQ ID NO: 192 (1 hr) 17.1 97SEQ ID NO: 192 (18 hr) 131.25 97 SEQ ID NO: 200 (1 hr) 32.1 97 SEQ IDNO: 200 (18 hr) 331.05 94 SEQ ID NO: 28 (1 hr) (200 μg) 32.1 100 SEQ IDNO: 28 (18 hr) (200 μg) 111.45 99 SEQ ID NO: 280 (1 hr) 0 n/a SEQ ID NO:280 (18 hr) 3.25 61 SEQ ID NO: 284 (1 hr) 2.3 100 SEQ ID NO: 284 (18 hr)55.35 98 SEQ ID NO: 172 (1 hr) 12.75 99 SEQ ID NO: 172 (18 hr) 205.9 96SEQ ID NO: 180 (1 hr) 38.7 93 SEQ ID NO: 180 (18 hr) 310.9 75 SEQ ID NO:168 (1 hr) 28 98 SEQ ID NO: 168 (18 hr) 301.1 90 SEQ ID NO: 228 (1 hr)39.2 99 SEQ ID NO: 228 (18 hr) 367 95 SEQ ID NO: 236 (1 hr) 14.85 96 SEQID NO: 236 (18 hr) 250.05 90 SEQ ID NO: 238 (1 hr) 30.05 97 SEQ ID NO:238 (18 hr) 466.15 90 SEQ ID NO: 240 (1 hr) 2.65 100 SEQ ID NO: 240 (18hr) 51.55 96 SEQ ID NO: 270 (1 hr) 12.2 91 SEQ ID NO: 270 (18 hr) 214.383 SEQ ID NO: 156 (1 hr) 62.5 88 SEQ ID NO: 156 (18 hr) 623.9 71 SEQ IDNO: 28 (1 hr) (50 μg) 31.3 98 SEQ ID NO: 28 (18 hr) (50 μg) 444.25 97

Total monatin production numbers ranged from undetectable to over 600ppm and % R,R ranged from 61% to 100%. Because the aminotransferase wasthe same for all of the aldolases, changing the aldolase can have asignificant impact on both the amount of monatin produced and thestereoisomeric distribution of the monatin produced.

The same experiments as above were done using L-tryptophan as thestarting substrate and coupling the aldolases (supplied as cellularextract) with HexAspC broad specificity L-aminotransferase produced andpurified as described in U.S. Published Application No. 20050282260 (0.5mg of purified protein). Results are shown below in Table 17 for totalmonatin production (subtracting background levels without aldolasepresent), and percent S,S monatin is shown based on reversed phase LCpeak area. Numbers above 400 ppm are outside the linear range of thestandard curve, and are approximate. Table 12 shows results for thebenchmark R-specific enzyme, the polypeptide with aldolase activity ofSEQ ID NO:28, which was assayed at the same time.

TABLE 17 Total monatin produced from L-tryptophan and % S,S % S,Smonatin Total (Reversed % S,S monatin monatin Phase (FDAA Aldolase (timepoint) (ppm) LC peak area) derivatization) SEQ ID NO: 278 (1 hr) 14.621.0 n/a SEQ ID NO: 278 (overnight) 7905 24.6 n/a SEQ ID NO: 162 (1 hr)14 15.4 n/a SEQ ID NO: 162 (overnight) 105.6 17.3 n/a SEQ ID NO: 276 (1hr) 35.8 10.2 n/a SEQ ID NO: 276 (overnight) 67.8 9 15.1 SEQ ID NO: 218(1 hr) 11.7 20.3 n/a SEQ ID NO: 218 (overnight) 49.9 17.7 22.0 SEQ IDNO: 244 (1 hr) 6.2 18.0 n/a SEQ ID NO: 244 (overnight) 24.4 14.7 19.6SEQ ID NO: 268 (1 hr) 6.3 24.1 n/a SEQ ID NO: 268 (overnight) 61.2 23.329.2 SEQ ID NO: 272 (1 hr) 5.7 20.5 n/a SEQ ID NO: 272 (overnight) 43.619.9 22.9 SEQ ID NO: 192 (1 hr) 6.8 19.0 n/a SEQ ID NO: 192 (overnight)56.4 20.0 24.4 SEQ ID NO: 172 (1 hr) 29.9 35.6 n/a SEQ ID NO: 172(overnight) 184.2 42.4 45.6 SEQ ID NO: 228 (1 hr) 59.6 23.9 n/a SEQ IDNO: 228 (overnight) 182 35.6 38.0

Example 13 Production of Monatin from Indole-3-Pyruvate Using aD-Aminotransferase

AT-103 transaminase was part of a transaminase library purchased fromBioCatalytics (Pasadena, Calif.) and the enzyme was tested forproduction of monatin in coupled reactions using the ProA aldolase fromC. testosteroni. The aldolase was prepared as described in WO 03/091396A2. AT-103 is a broad specificity D-transaminase (E.C. 2.6.1.21) from aBacillus species that requires a D-amino acid (such as D-glutamate,D-aspartate, or D-alanine) as the amino acid donor. Enzymes andadditional components/substrates were added directly to the reactionbuffer provided in the kit, which contained 100 mM potassium phosphatebuffer pH 7.5, 100 mM amino donor, and 0.1 mM PLP. To one mL of reactionbuffer were added: 4 mg indole-3-pyruvate, 20 mg pyruvate, approximately50 μg ProA provided in a cellular extract, 1 μL 2 M MgCl₂, and 2 mg ofaminotransferase enzyme. Reactions were performed in duplicate. Thereactions were incubated overnight at 30° C. with gentle shaking (100rpm). The samples were filtered and submitted for reversed-phaseLC/MS/MS analysis as described in Example 1. The results indicated thatapproximately 370 μg/mL monatin were produced using AT-103 enzyme. Theresults were further analyzed to determine ratios of S,R/R,S versusR,R/S,S monatin, on the basis of the peak areas of the two stereoisomerpools that resolve during the chromatographic separation. Of the totalmonatin produced by AT-103, 69% was R,R/S,S monatin in comparison to themixed isomers. This enzyme is homologous to the Bacillus subtilis DATenzyme described in WO 03/091396 A2, which is known to have a broadspecificity for D-amino acids. Chiral analysis was performed using theFDAA methodology described in Example 1, which verified that theD-aminotransferase was making predominantly R,R monatin, and some S,Rmonatin as expected. Further transamination experiments with S,S monatinor R,R monatin and α-ketoglutarate as substrates verified that theBioCatalytics enzyme was highly selective for the D-configuration atcarbon 4, as expected. In these experiments, no glutamate was detectedin the reaction with S,S monatin and α-ketoglutarate as substrates.

To decrease the amount of S,S monatin or R,S monatin produced asbyproducts in coupled reactions with AT-103 (the broad rangeD-transaminase) and the ProA aldolase, the aldolase was purified usingHis-Bind cartridges, following manufacturer's protocols (Novagen,Madison, Wis.). The purified enzyme preferably should not containwildtype L-aminotransferase activities that can be present in cellularextracts (such as the native E. coli AspC or TyrB activities). TheHis-Bind eluent was desalted to remove imidazole using PD-10 columns(G25 Sephadex, GE Healthcare, Piscataway, N.J.) and was eluted in 50 mMTris-Cl, pH 7. Experiments were carried out in duplicate in a volume of1 mL and contained 100 mM Tris-Cl buffer, pH 7.8, 50 μg ProA aldolase, 4mg indole-3-pyruvate, 1 or 2 mg D-aminotransferase, 200 mM sodiumpyruvate, 2 mM MgCl₂, 3 mM potassium phosphate, 0.1 mM PLP, and 14.7 mgof D-glutamate. The tubes were incubated at 30° C. with gentle shaking.Two-hour time points were taken and frozen immediately at −20° C. The pHwas adjusted at two hours from 5 to between 7-8 using NaOH, and theassays were incubated overnight. Samples were filtered and analyzed formonatin as described in Example 1. The two-hour samples did not havedetectable amounts of monatin, probably due to the low pH. The overnightsamples contained approximately 190 ng/mL monatin when 1 mg ofD-aminotransferase was used, and approximately 84% was R,R monatin and16% was S,R monatin. When 2 mg of D-aminotransferase were used, 540ng/mL monatin was produced, approximately 71% was R,R monatin.

Similar experiments were conducted using BioCatalytics Aminotransferasebuffer (BioCatalytics, Pasadena, Calif.), which contained 100 mMpotassium phosphate pH 7.5, 0.1 mM PLP, and 100 mM D-glutamate. Solidindole-3-pyruvate and D-aminotransferase were added as above. ProAaldolase (50 μg), MgCl₂, and 50 mM pyruvate were added from stocksolutions. The assays were treated as above, although no pH adjustmentwas required in this case. A negative control was done with just theBioCatalytics supplied enzyme and buffer, which did not contain monatin.The experimental results are shown in Table 18.

TABLE 18 Production of Monatin from Indole-3-Pyruvate in PhosphateBuffer Mg D- Total Monatin aminotransferase Time (hrs) (ng/mL) % R,R 0 20 n/a 1 2 6780 not determined 2 2 13170 55% 0 16 0 n/a 1 16 15000 notdetermined 2 16 28930 51%

The production of monatin in phosphate buffer is clearly higher thanthat in Tris buffered systems.

To compare activities of the cloned B. subtilis DAT from WO 03/091396 A2with the BioCatalytics enzyme (AT-103) (BioCatalytics, Pasadena,Calif.), additional assays were done. The B. subtilis dat gene was alsosubcloned into pET30a to remove the His-6 tag. Untagged and taggedenzyme were produced in BL21(DE3), as described in WO 03/091396 A2.Cellular extracts were made and total protein assays were done toestimate protein concentration as described previously. Duplicate one mLreactions were done which contained: 500 μg D-aminotransferase, 50 μgProA aldolase, 100 mM potassium phosphate pH 7.5, 3 mM MgCl₂, 4 mgindole-3-pyruvate, 200 mM sodium pyruvate, 7.35 mg (50 mM) D-glutamate,and 0.1 mM PLP. Samples were incubated at 30° C. for 1 hr, 2 hr, andovernight, and were filtered for LC/MS/MS analysis. The samplescontained only the S,R and R,R stereoisomers of monatin, as determinedby the FDAA derivitization protocol described in Example 1. The resultsare summarized in Table 19 below. The % RR was determined by peak areasthat were separated by reversed phase chromatography.

TABLE 19 Comparison of D-aminotransferase enzymes Enzyme Time (hr)Monatin (ppb) % RR monatin B. sub DAT-HIS 1 512 not determined B. subDAT untagged 1 1056 not determined BioCatalytics AT-103 1 2353 notdetermined B. sub DAT-HIS 2 894 ~80-90% B. sub DAT untagged 2 1913  ~80%BioCatalytics AT-103 2 6887 92.5% B. sub DAT-HIS 16 3014 31 B. sub DATuntagged 16 5612 33 BioCatalytics AT-103 16 16131 66

The removal of the HIS-6 tag appears to have improved the activity ofthe B. subtilis D-aminotransferase; however, the BioCatalyticsD-aminotransferase homolog clearly had the highest activity. TheBioCatalytics D-aminotransferase homolog also showed greater substratespecificity for the R-monatin precursor. Increased incubation timesappear to reduce the enantiomeric excess of R,R monatin that isproduced.

Because the Bacillus D-aminotransferase enzymes have a preference forpyruvate as an amino acceptor and D-alanine as an amino donor. It wasexpected that D-alanine could be utilized as the amino donor forconversion of MP to monatin with similar or better results. Duplicateone mL reactions were done which contained: 500 μg D-aminotransferase,50 μg purified ProA aldolase, 100 mM potassium phosphate pH 7.5, 3 mMMgCl₂, 4 mg indole-3-pyruvate, 100 mM sodium pyruvate, 25 mM D-glutamateor D-alanine, and 0.1 mM PLP. Samples were incubated for 2 hours, andtreated as above prior to analysis. When D-alanine was used as the aminodonor, slightly higher levels of monatin were produced (23 versus 21ppm) as expected. Additionally, it is expected that high concentrationsof pyruvate may inhibit the transamination step, thus dosing in smalleramounts of pyruvate over time may improve the overall rate of monatinproduction. One can see from the above data that even though one-half ofthe pyruvate was used in this case compared to the above table,significantly more monatin was produced. AT-103 is an example of anenzyme with limited activity for S-MP. Even though the ProA aldolaseused in this study makes greater than 90-95% S-MP, AT-103 makes up to92% R,R monatin.

Example 14 Production of R,R Monatin from D-Tryptophan

The following were added per 1 mL of reaction mixture: approximately 60μg C. testosteroni ProA aldolase (supplied in cellular extracts, asdescribed in WO 03/091396 A2), 4 mM MgCl₂, 50 mM D-tryptophan, 0.5 mgBioCatalytics D-aminotransferase (AT-103) (BioCatalytics, Pasadena,Calif.), 100 mM sodium pyruvate, 100 mM potassium phosphate buffer pH7.5 or 100 mM sodium acetate buffer pH 8, 0.05 mM PLP, 3 mM potassiumphosphate (only to the acetate reactions), and 10 mM α-ketoglutarate.Experiments were run in duplicate, with negative controls in which noaldolase was added. Samples were incubated overnight (20 hours) at 30°C. with gentle shaking. The actual pH of the sodium acetate samples wasapproximately 5, while the final pH for the phosphate buffered sampleswas approximately 7. None of the aldolases appeared to have significantactivity at pH 5, the sample containing ProA aldolase was slightly abovethe negative control but probably not above experimental error. Inpotassium phosphate, the ProA aldolase produced 73.4 ppm monatin with aratio of R,R:S,R of 1.7:1 (˜63% R,R from D-tryptophan).

Because the Bacillus D-aminotransferase enzymes have a preference forpyruvate as an amino acceptor and D-alanine as an amino donor, it wasexpected that the addition of alpha-ketoglutarate is unnecessary whenproducing R,R or S,R monatin from D-tryptophan. The above experiment wasrepeated (in 100 mM potassium phosphate buffer) using purified ProAaldolase (50-60 μg), and an incubation time of 2.5 hours. Duplicateexperiments were run, with and without alpha-ketoglutarate. With 10 mMalpha-ketoglutarate added, 56.1 ppm monatin was formed from D-tryptophan(79.5% R,R, 20.5% S,R). Without alpha-ketoglutarate, 102.5 ppm monatinwas formed (79% R,R, 21% S,R).

Example 15 Tryptophan Racemase

R,R-monatin has been produced using D-aminotransferase and an aldolasewhen D-tryptophan was used as the starting material (Example 14). Thatnotwithstanding, L-tryptophan may be a preferred starting material forseveral reasons. For example, L-tryptophan may be less expensive andmore readily available than D-tryptophan. This disclosure describesseveral methods for obtaining an active tryptophan racemase. Yields ofR,R monatin are improved by using an R-specific aldolase, i.e., analdolase that preferentially or selectively produces R-MP. FIG. 3illustrates a method for producing stereoisomerically-enriched R,Rmonatin from L-tryptophan using a tryptophan racemase, aD-aminotransferase and an R-specific aldolase.

A selection for a tryptophan racemase is created by constructing astrain that would require an active racemase for growth. A tryptophanauxotroph needs a source of L-tryptophan when grown on minimal medium.Supplementing the medium with D-tryptophan is one way to select for aracemase that converts D-tryptophan to L-tryptophan. The tryptophanauxotrophs were tested for growth on minimal medium supplemented withD-tryptophan. The strains, CAG18455 and CAG18579 from the Coli GeneticStock Center and NRRL12264 (Also lipA⁻, λDE3lysogenized, and cured ofits plasmid), did not grow when supplemented with D-tryptophan but grewwhen supplemented with L-tryptophan. E. coli may be used as a hostorganism but other host organisms also may used, such as yeast, otherbacteria, or other eukaryotic organisms. A tryptophan auxotroph(specifically NRRL12264 (also lipA⁻, λDE3 lysogenized and cured of itsplasmid)) will grow on D-tryptophan when it has been transformed with aD-aminotransferase. This confirms the ability of E. coli to transportD-tryptophan into the cell.

Salcher and Lingens described the presence of a tryptophan racemase inPseudomonas aurereofaciens (ATCC15926). Tryptophan racemase has alsobeen described in several plants including tobacco, beets, tomato, andwheat and the enzyme appears to be induced by conditions of osmoticstress or drought. Tryptophan racemase may play a role in Sclerochitonilicifolius in the native monatin production pathway. To isolate thisracemase activity, an expression library is constructed from ATCC15926(or another organism with tryptophan racemase activity) and the libraryis transformed into the tryptophan auxotroph. A strain is selected thatwill grow using D-tryptophan as the tryptophan source. A similar methodis also used to screen many strains with known racemases to look for aracemase with activity on D-tryptophan. Examples include: alanine,serine, and glutamate racemases (T. Yoshimura and N. Esaki, “Amino AcidRacemases: Functions and Mechanisms.” Journal of Bioscience andBioengineering, Vol. 96, No. 2, 103-109, 2003). Alanine racemase is PLPdependent and has been cloned from Salmonella typhimurium (dadB gene).Other sources are Escherichia coli, Bacillus subtilis, Pseudomonasaeruginosa, Vibrio cholerae, Schizosaccaroyces pombe, and Bacilluscereus. A basidiomycetous mushroom, Lentinus edodes, also contains abroad activity alanine racemase. Serine racemase is also PLP dependentand is found in Eukaryotes (such as silkworm, rat brain, mouse braincDNA) as well as bacteria (Enterococcus gallinarum). Glutamate racemaseis PLP-independent and has been cloned from Pediococcus pentosaceus,Bacillus pumilus, Lactobacillus fermenti, Lactobacillus brevis, E. coli,Aquifex pyrophilus, and Bacillus subtilis. The glutamate racemase isvery specific and, consequently, even structurally similar amino acidsaspartate, asparagine, and glutamine are not substrates for the enzyme.Aspartate racemases also exist and are PLP independent. These enzymesare found in Lactobacilli, Streptococcus strains, and some archaea suchas Desulfurococcus and Thermococcus strains. The bivalve molluskScapharca brouhtonii also contains an aspartate racemase. Otherracemases found in the literature include amino acid racemase (EC5.1.1.10) from Anabaena sp. and Pseudomonas striata, proline racemase,multifunctional phenylalanine racemase. Related epimerases or racemasesare also being tested. Potential racemases are tested to make sure theyare not D-tryptophan aminotransferases. This screening is done bysequence analysis and/or an enzyme assay.

Enzymes that pass the test as a racemase are screened for activity onmonatin as described in Example 17. Ideally, one obtains an enzyme thatis very specific for tryptophan and has little or no racemase activityon monatin.

A tryptophan racemase also may be evolved and/or improved (viamutagenesis or recombinant engineering) from an existing racemase,transaminase, or epimerase. Additionally, because crystal structures foralanine aminotransferases are known, these may be used as a basis forrational, structure based mutagenesis. The process described above isused as an initial selection for tryptophan racemase activity and as ascreen for improved activity.

Tryptophan Racemase Libraries

Construction of Libraries:

Burkholderia pyrrocina (ATCC15958) and Pseudomonas chlororaphis(ATCC15926) were obtained from the American Type Culture Collection.They were grown as recommended by ATCC and genomic DNA was preparedaccording to the method of Mekalanos J J. (Duplication and amplificationof toxin genes in Vibrio cholerae. Cell. 1983. 35:253-63). The genomicDNA was partially digested with the Sau3AI restriction enzyme. 1-3 Kbpfragments were gel purified using a Qiagen QIAquick Gel Extraction Kit(Qiagen, Valencia, Calif.). The purified DNA was ligated into pTrc99a(Amersham, Piscataway, N.J.) that had been digested with BamHI andpurified as above. The ligation was done at room temperature withovernight incubation using a 3:1 molar ratio of insert to vector. Theligated library was transformed into TOP10F′ chemically competent cells(Invitrogen, Carlsbad, Calif.) and plated on LB medium with 100 μg/mlampicillin. After overnight incubation of the transformation plates,colonies were scraped off of the plates washed with liquid LB medium andan appropriate size cell pellet was mini-prepped using a Qiagen QIAquickmini-prep kit (Qiagen, Valencia, Calif.). Approximately 30,000 colonieswere pooled and mini-prepped.

The pooled plasmid was transformed into CAG18455 (trpC83::Tn10, rph-1)or CAG18579 (trpC::Tn10kan, rph-1). Both strains are tryptophanauxotrophs so they will not grow on M9 minimal medium (Difco) unless themedium is supplemented with tryptophan. The transformants were plated onM9 minimal medium supplemented with D-tryptophan. This selects for astrain that can convert D-tryptophan to L-tryptophan.

Prior to transformation of the library, the strains were tested forgrowth on minimal medium with L- or D-tryptophan. The strains weretested for growth on minimal medium supplemented with D-tryptophan andno growth was observed. Both strains grew on identical mediumsupplemented with L-tryptophan instead of D-tryptophan. Additionally, aderivative of NRRL12264 (the strain used had been cured of thetryptophan operon plasmid, lysogenized with λDE3, and deleted for lipA,in addition to the other chromosomally encoded mutations (serB, ΔtrpED,tnaA2, aroP) (this strain could not grow on minimal medium supplementedwith D-tryptophan but grew on identical medium supplemented withL-tryptophan instead of D-tryptophan) was transformed with a D specificaminotransferase from Bacillus subtilis (WO 03/091396). Expression ofthe D-aminotransferase was driven by the T7 promoter. The transformedstrain was able to grow on M9 minimal medium supplemented withD-tryptophan.

The colonies that grow on the D-tryptophan medium are screened. Theplasmid is isolated and retransformed into the parent strain (CAG18455or CAG18579) to confirm that growth on D-tryptophan medium is dependenton the plasmid and not on a host mutation. The nucleotide sequences ofthe plasmids that complement the tryptophan auxotrophy are analyzed.Clones that are determined to contain a tryptophan racemase gene arefurther analyzed.

The tryptophan racemase from other tissue sources is isolated in asimilar fashion. There are literature reports of tryptophan racemaseactivity in both tobacco tissue culture cells (Nicotiana tabacum L. var.Wisconsin 38) (Miura, G. A. and Mills, S. E. The conversion ofD-tryptophan to L-tryptophan in cell cultures of tobacco. Plant Physiol.1971. 47:483-487) and in crude protein extracts of wheat (Triticumaestivum) (Rekoslayskaya, N. I., Yur'eve, O. V., Shibanova, L. A., andSalyaev, R. K. Synthesis and physiological function of D-tryptophanduring wheat germination. Russian J. Plant Physiol. 1997. 44:196-203). AcDNA expression library is made from tissue, as described in theliterature, and the expression library is used to transform a tryptophanauxotroph as described above.

Tryptophan Racemase Assay

Clones that are identified as potentially having a tryptophan racemaseare transformed into a strain of E. coli commonly used for expression ofrecombinant proteins, such as BL21. The cells are grown in LB broth toan optical density at 600 nm of 0.4-0.6. The promoter driving expressionof the racemase is induced with IPTG (0.1 mM final concentration). Afterinduction, the cells are allowed to express the protein for 1-3 hours at37° C. (with aeration). The cells are harvested and lysed by Frenchpress, sonication, or by chemical means (such as BugBuster (Novagen,Madison, Wis.)). The lysed cells are centrifuged to remove the celldebris. The clarified extract is used directly in assays.

Varying amounts of extract is added to a solution such that the finalconcentration is 50 mM potassium phosphate (pH 7.0) and 2 mML-tryptophan. Pyridoxal-5′-phosphate is added at a final concentrationof 10 μM. The samples are incubated and then analyzed by LC/MS. Thepresence of a D-tryptophan peak when only L-tryptophan is used as asubstrate indicates a positive result. D-tryptophan concentration shouldincrease with increasing time until equilibrium is reached, and the rateshould also increase with protein concentration until the concentrationof enzyme is high enough that it is no longer saturated with substrate.D-tryptophan may also be converted to L-tryptophan as above.

A complementing gene may code for a D-aminotransferase. (A“complementing gene” is a gene that, when expressed, nullifies amutation in an organism. For example, if an organism has a null mutationin one of the genes required for synthesis of tryptophan by the cell, acomplementing gene could be one that, when expressed, allows the strainto grow on minimal medium (i.e., without tryptophan). This reactionrequires an alpha-keto acid such as α-ketoglutarate, oxaloacetate, orpyruvate as an amino acceptor. These compounds will likely be present ina cell extract (in small amounts). These compounds may be removed usinga PD-10 desalting column (GE Healthcare, Piscataway, N.J.) and the assaymay still be performed in crude extract. The tryptophan racemaseactivity is purified using conventional column chromatography. Finally,the open reading frame identified as a potential tryptophan racemase iscloned into an expression vector with an affinity tag. The potentialtryptophan racemase is then purified by affinity chromatography. Ineither case the purified protein is used in enzyme assays essentially asdescribed above.

Reverse Genetic Engineering of Tryptophan Racemase

The tryptophan racemase may be purified from either plant or microbialsources by conventional protein purification techniques includingammonium sulfate fractionation, and conventional column chromatography.Once the protein has been purified such that a spot can be isolated on a2-D gel, peptide microsequencing techniques or conventional Edman typeamino acid sequencing are utilized (see golgi.harvard.edu/microchem/fordescriptions of the protocols and equipment used for this type of work).In some cases, however, the genome sequence of the organism cannot beused as a source of the protein for the protein purification becausesuch sequence has not been determined yet. In that situation, the firstset of degenerate primers may be designed based on sequence availablefrom the closest known relative of the protein source. Degenerate PCRand genome walking is then be performed according to establishedprotocols to isolate the tryptophan racemase coding sequence.

Tryptophan Racemase Monatin Production

The following is added per 1 mL of reaction mixture: approximately 60 μgC. testosteroni ProA aldolase (supplied in cellular extracts, asdescribed in WO 03/091396 A2), 100 μL/mL of tryptophan racemase cellularextract or 1 mg/mL purified tryptophan racemase, 4 mM MgCl₂, 50 mML-tryptophan, 0.5 mg BioCatalytics D-aminotransferase (AT-103)(BioCatalytics, Pasadena, Calif.), 100 mM sodium pyruvate, 100 mMpotassium phosphate buffer pH 7.5, 0.05 mM PLP, and 10 mMα-ketoglutarate. Because pyruvate is an acceptable amino acceptor forthe broad specificity D-aminotransferase, α-ketoglutarate is optional.Experiments are run in duplicate, with negative controls in which noaldolase was added or no tryptophan racemase was added. Samples areincubated˜1 hour or overnight (20 hours) at 30° C. with gentle shaking.

The tryptophan racemase is tested for activity on monatin. An assaysimilar to that in Example 17 is used with monatin as a substrate, andcompared to the activity of the enzyme on tryptophan. The ideal enzymehas activity on tryptophan but little or no activity on other aminoacids particularly glutamate and monatin. If the enzyme has significantactivity on monatin, the enzyme may be mutagenized to decrease theactivity on monatin and or glutamate while keeping the tryptophanactivity unchanged or at a level high enough for the enzyme to be usefulin monatin production. Techniques that may be used for mutagenesisinclude, but are not limited to, error prone PCR, site-directedmutagenesis, modeling to identify site-directed mutagenesis targets(sites that may be involved in substrate binding), passage throughmutagenic strains, and DNA shuffling.

Mutagenized racemases may be screened for tryptophan activity using aplate assay as described above. Clones that retain tryptophan activityare then screened for a loss of activity on monatin.

Example 16 Site Directed Mutagenesis of HEXAspC Experimental Overview

A hexamutant of E. coli AspC (HEXaspC) was found to have better activityas compared to AspC for the production of S,S monatin as described inExample 6 of WO 03/091396 A2. HEX (accession number:/AHFA gi:127190)contains the following mutations from AspC (E. coli numbering): V35L,K37Y, T43I, N64L, T104S, and N285S. Based on structural analysis andliterature reports (S. Rothman and J. Kirsch, J. Mol. Biol. (2003), 327,593-608; S. Rothman et al., Protein Science (2004), 13: 763-772), 5 moremutants were created that were expected to increase the kinetic activitytoward substrates utilized in the monatin production pathway:L-tryptophan, S-MP, or both. Two of the mutants increased transaminationrates for both tryptophan and S,S monatin. Two of the mutants showed anincreased stereoselectivity for the formation of S,S monatin while onewas less stereoselective. Based on this, it is expected that the broadspecificity D-aminotransferase from Bacillus sp. having similarmutations is useful as the D-aminotransferase in the R,R monatinpathways shown in FIG. 3, and described in Example 15. One of themutants (HEXaspCP9T/R122G) had increased activity for L-tryptophantransamination, but activity in S,S monatin production or S,S monatintransamination was decreased significantly. Thus, it is expected thatthis enzyme is useful in the first step of the R,R monatin productionpathways shown in FIGS. 1, 2, 4, 5, 6, 7, and 8 and described inExamples 18 and 19. In general, an aminotransferase that has activitysimilar to that of AspC on L-tryptophan and limited activity on R-MP andS-MP would be useful for the processes depicted in FIGS. 1, 2, 4, 5, 6,7, and 8.

Methods and Materials

The HEX gene cloned in pUC19 was provided by Professor J. F. Kirsch(Department of Molecular and Cell Biology, University of California,Berkeley, Berkeley, Calif. 94720-3206) and used as the template for thecloning of the gene into pET23a. See James J. Onuffer and Jack F.Kirsch, Redesign of the substrate specificity of Escherichia coliaspartate aminotransferase to that of Escherichia coli tyrosineaminotransferase by homology modeling and site-directed mutagenesis,Protein Science, 4: 1750-1757 (1995). See also NCBI accession number1AHF_A GI:1127190 (HEX amino acid sequence). The following primers weredesigned for cloning the HEX gene into the pET23a vector (Novagen,Madison, Wis.):

HEXaspC primers: N term: (SEQ ID NO: 393)5′-GCGGAACATATGTTTGAGAACATTACCGCC-3′; C term: (SEQ ID NO: 394)5′-ATAACCGGATCCTTACAGCACTGCCACAATCG-3′

The following PCR protocol was used for gene amplification: In a 100 μLreaction, 50 ng DNA template, 1.0 μM of each primer, 0.2 mM each dNTP, 1U Pfu Turbo Polymerase (Stratagene, La Jolla, Calif.), and 1× Cloned Pfubuffer were added. The thermocycler program utilized a hot start of 94°C. for 5 minutes; followed by 25 cycles of a denaturing step at 94° C.(30 sec), an annealing step at 55° C. (1 min), and an extension step at72° C. (2 min) and finally a finishing step at 72° C. (7 min). Standardmolecular biology techniques were utilized to clone the PCR product intopET23a using NdeI and BamHI restriction sites.

The tryptophan residue at position 130 is thought to be important forstacking interactions with the pyridoxyl ring, but also appears to be asource of steric hindrance with the S-monatin precursor (MP) substrate,based on protein modeling observations. Therefore, an amino acid with asmaller hydrophobic side chain (phenylalanine) was used to replace thetryptophan. The rest of the mutations were based on kinetics data inliterature, although new combinations of desirable mutations werecreated. All mutations to HEXaspC, with the exception of W130F, weremade using the Stratagene Multi-Change kit (Stratagene, La Jolla,Calif.) by following the manufacturer's instructions. The W130F mutationwas made using the Stratagene QuikChange kit (Stratagene, La Jolla,Calif.) according to the manufacturer's instructions with the onlyexception being that the extension temperature for the PCR reaction wasdecreased to 66° C. The primers for the multi-change kit were designedusing the QuikChange multi-kit primer design tool on<www.stratagene.com>, except for the W130F single mutation primers.

The primer sequences are listed below in Table 20.

TABLE 20 Primer Sequence (5′ to 3′) aspCW130F_backwardCGCTCTTATGGTTCGGTTTGCTTGGGTTGCTC ACCC (SEQ ID NO: 395) aspCW130F_forwardGGGTGAGCAACCCAAGCTTTCCGAACCATAAG AGCG (SEQ ID NO: 396) R122G-1^(a)CAAAAAATACCAGCGTTAAGGGAGTGTGGG TGAGCAACC (SEQ ID NO: 397) P9T_4^(a)CATTACCGCCGCTACTGCCGACCCGATTC (SEQ ID NO: 398) I68V-1^(a)CACCAAAAATTACCTCGGCGTAGACGGCAT CCCTGAATT (SEQ ID NO: 399) T156A^(a)TGATGCGGAAAATCACGCTCTTGACTTCGATG CAC (SEQ ID NO: 400) ^(a)Denotes aprimer that was modified by 5′ phosphorylation

Expression of HEXaspC Mutant Genes and Analysis of Enzyme Activity

Liquid cultures (5 mL) of Novagen Overnight Express™ AutoinductionSystem 2 (Catalog #71366-3; solutions 1-6) (Novagen, Madison, Wis.) wereinoculated from fresh plates or frozen glycerol stocks of the followingstrains:

E. coli BL21(DE3)::HEXaspCpET23aE. coli BL21(DE3)::HEXaspCW130FpET23aE. coli BL21(DE3)::HEXaspCT156ApET23aE. coli BL21(DE3)::HEXaspCP9T/T156ApET23aE. coli BL21(DE3)::HEXaspCP9T/R122GpET23aE. coli BL21(DE3)::HEXaspCR122G/T156ApET23a.

The cultures were incubated at 37° C. at 230 rpm for 6-8 h. The OD₆₀₀ ofeach culture was determined and the volume of culture necessary toobtain an OD₆₀₀ of 0.03-0.05 in 25 mL was calculated. The calculatedvolumes of each liquid culture were transferred to flasks containing 25mL of the same medium. The Overnight Express™ Autoinduction System 2 isa complete, chemically defined medium for high-level expression withIPTG-inducible expression systems that uses lactose as the inducingagent and does not require monitoring of cell growth. The OvernightExpress cultures were incubated at 30° C. with shaking at 230 rpm for 18h. The cells were harvested by centrifugation and washed once with cold50 mM MOPS, pH 7.0. The cells were then lysed using Bugbuster™ (primaryamine free) Extraction Reagent (Novagen Catalog #70923-3) (Novagen,Madison, Wis.) containing 1 μL/mL benzonase nuclease (Novagen Catalog#70746-3) (Novagen, Madison, Wis.), 5 μL/mL Protease Inhibitor CocktailSet II (Novagen Catalog #539132) (Novagen, Madison, Wis.) and 0.33 μL/10mL r-Lysozyme (Novagen Catalog #71110-3) (Novagen, Madison, Wis.)following the Novagen recommended protocol. After incubation at 25° C.for 15 minutes with gentle shaking, the cell debris from each suspensionwas pelleted by centrifugation at 21,000 g for 15 minutes at 4° C. Thesupernatant was carefully decanted and analyzed as the cell freeextract. Inclusion body fractions were isolated by suspending the celldebris fractions in 30% Bugbuster™ (primary amine free) ExtractionReagent, centrifuging at 21,000×g for 10 min; suspending the centrifugedpellets in 10% Bugbuster™ (primary amine free) Extraction Reagent,centrifuging again to isolate the washed pellets. The cell free extractsand inclusion body fractions were analyzed for protein expression bySDS-PAGE on 4-15% gradient gels (BioRad #161-1104, Hercules, Calif.).For the cell extract samples, twenty micrograms of soluble protein wereloaded in each gel lane (premixed with 1× protein loading buffer andheated at 95 C. for 5 min). The inclusion body fractions were dissolvedin 1× protein loading buffer (0.2 mL), heated for 10 minutes at 95 C.and 5 μL of each solution was loaded per gel lane. The amount of eachHEX mutant in comparison to the total soluble protein loaded into eachlane was calculated by band intensity analysis using Labworks BioImaging1D-gel tool (UVP, Inc. Upland, Calif.), and is reported below:

TABLE 21 HEXaspC protein/total soluble Sample protein E. coliBL21(DE3)::HEXaspCP9T/T156ApET23a CFE 0.310 E. coliBL21(DE3)::HEXaspCP9T/R122ApET23a CFE 0.145 E. coliBL21(DE3)::HEXaspCpET23a CFE 0.172 E. coliBL21(DE3)::HEXaspCR122A/T156ApET23a CFE 0.174 E. coliBL21(DE3)::HEXaspCW130FpET23a CFE 0.114 E. coliBL21(DE3)::HEXaspCT156ApET23a CFE 0.120

Analysis of the gels showed that the HEXaspCR122A/T156A mutant was theonly protein that was found in substantial quantities as inclusionbodies. The HEXaspCP9T/T156A protein gave the highest level ofexpression, approximately 90% better than HEXaspC protein. In contrast,the W130F, T156A and P9T/R122G proteins were expressed in lowerconcentrations than HEXaspC.

The activity of the HEXaspC mutant proteins for the production ofS,S-monatin was measured using the following reaction conditions: Each 1mL reaction contained 50 mM TAPS, pH 8.2, 4 mM MgCl₂, 3 mM sodiumphosphate, pH 8.0, 200 mM sodium pyruvate (pH adjusted to 8), 5 mMα-ketoglutarate (pH adjusted to 8), 50 mM tryptophan, 0.05 mM pyridoxal3-phosphate, 50 μg/mL ProA aldolase (added as a cell free extract) andvarying concentrations (approximately 50 and 500 μg/mL) ofaminotransferase (added as a cell free extract). Deaerated water wasused to prepare the stock solutions and to adjust the volume of thereaction mixtures to 1.0 mL. The pyridoxal phosphate was added justprior to the addition of the enzymes. The reaction tubes were incubatedat 30° C. with gentle shaking for 4 h. Samples (0.01 mL) were withdrawnat 1, 2, and 4 h after the addition of the enzymes, filtered, andanalyzed by LC/MS/MS, as described in Example 1. Monatin production wasnormalized based on the amount of aminotransferase present in thereactions. Under the conditions of these assays, the HEXaspC and theHEXaspCT156A produced the most total monatin per mg of aminotransferasewhile the P9T/R122G protein produced the least, followed byHEXaspCW130F. The HEXaspCW130F and P9T/R122G enzymes showed the greateststereoselectivity for S-MP (greater than 98% S,S-monatin), even whenhigh enzyme concentrations were used (greater than 300 μg/mL). Thepercentage of S,S-monatin product decreased to less than 90% in theenzymatic reactions containing the P9T/T156A enzyme at highconcentration. The other mutants showed a product stereoselectivity verysimilar to the original HEXaspC mutant (approximately 95% S,S-monatin).Analysis of the product of the reaction containing the HEXaspC enzymeusing the FDAA derivitazation reagent described in Example 1 showed thatthe second stereoisomer formed is R,S-monatin.

Assaying of Tryptophan and Monatin Aminotransferase Activity

The mutants were tested for transamination activity using S,S monatinand L-tryptophan as substrates. The aminotransferase activity wasmeasured by following the formation of the co-product of the reaction,glutamate, by HPLC with OPA-post-column derivitization as described inExample 1. The reaction mixture contained, in 1.0 mL, 100 mM HEPPSbuffer, pH 8.0, 20 mM alpha-ketoglutarate, 0.08 mM pyridoxal phosphate,25 mM tryptophan or S,S monatin, and enzyme (supplied as 2.5 mg of incellular extracts protein). All components except the enzyme were mixedtogether, the enzyme was added to start the reaction and the reactionsolution was incubated at 30° C. (gentle shaking) for 90 minutes.Reactions were done in duplicate, with negative controls in which noenzyme was added. The reaction was stopped by the addition of 10% formicacid (final concentration), the mixture was centrifuged at 21,000 rpm,and the supernatant was carefully removed and filtered. The data werecorrected for background levels of glutamate and for the dilution fromthe addition of acid to precipitate the proteins, then normalized byamount of mutant aminotransferase added. When tryptophan was utilized asa substrate, HEXaspC produced 13.0 mM glutamate per mg ofaminotransferase per hour. The relative activity, expressed as apercentage, of the mutants is as follows: HEXaspCW130F (156%),HEXaspCT156A (151%), HEXaspCP9T/T156A (63.7%), HEXaspCP9T/R122G (116%),and HEXaspCR122G/T156A (107%). When S,S monatin was utilized as asubstrate, HEXaspC produced 7.43 mM glutamate per mg of aminotransferaseper hour. The relative activity, expressed as a percentage, of themutants is as follows: HEXaspCW130F (113%), HEXaspCT156A (87.7%),HEXaspCP9T/T156A (67.3%), HEXaspCP9T/R122G (11.2%), andHEXaspCR122G/T156A (114%).

The HEXaspCP9T/R122G mutant had increased activity for tryptophan toindole-3-pyruvate conversion, but decreased activity for S,S monatintransamination. The ratio of tryptophan to monatin activity was 18.2 incomparison to 1.75 for HEXaspC, making it a desirable candidate forproduction of R,R monatin using pathways that require anL-aminotransferase such as those described in Examples 18 and 19. Assuch, the HEXaspCP9T/R122G is an example of an aminotransferase withlimited activity on S,S monatin (and MP).

Most of the mutations improved L-tryptophan activity, but only twomutants increase activity toward both L-tryptophan and S,S monatin(HEXaspCW130F and HEXaspCR122G/T156A). Because 25 mM of substrate wasused in these assays, the enzymes were most likely saturated and theactivity is a reflection of the k_(cat) of the enzymes. However, underthe conditions in which the assays for S,S monatin production wereperformed (above) it is unlikely that the concentration of S-MP issufficient to saturate the enzyme, thus the increase in k_(cat) is notreflected. It has been reported, for similar substrates, that some ofthe mutations made increase the k_(cat) but also increase the apparentK_(m) for the amino acid substrate. If increasing concentrations ofsubstrates were used, it is expected that these two mutants wouldprovide a benefit in production rates of S,S monatin in comparison toHEXaspC. The HEXaspCT156A mutation appears to have increased tryptophantransamination rates without having a significant effect on MPtransamination rate under the conditions above for S,S monatinproduction.

By comparison of the structures of HEXaspC and one of the Bacillus sp.D-aminotransferase enzymes (see, for example, S. Sugio, G. A. Petsko, J.M. Manning, K. Soda, and D. Ringe, Biochemistry 34 (1995) pp.9661-9669), the W130F, R122G, T156A, and HEX mutations of AspC could bemapped to corresponding residues in the D-aminotransferase structure. Itis expected that similar mutations in the broad specificityD-aminotransferase would improve the overall production of R,R monatinas described in Example 14. For example, the functionality provided bytryptophan 130 in AspC is replaced in Bacillus D-aminotransferases byhydrogen bonding between the side chains of serines 179-181 andglutamate 166 (YM-1 numbering scheme). To lessen steric hindrance, theglutamate could be mutated to an aspartate residue. SomeD-aminotransferases have a threonine residue at position 179, whichwould increase steric hindrance and should be avoided. The B. sphearicusenzyme has an alanine in place of serine at position 181, which may alsoreduce steric hindrance.

Additional information from studies of aspartate aminotransferase can beapplied to the D-aminotransferase as well. While the AspC enzyme has anarginine in the active site that interacts with the side chain ofdicarboxylate substrates, the D-aminotransferase has a loop from Ser240to Ser243. The side chains of Ser240, Thr242, and Ser243 face the samedirection and form a pocket with the hydroxyl group of Ser180 whichprovides a surface for both nonpolar and polar substrates can interact.Ser180 is involved in PLP binding; however, to improve the activity of aD-aminotransferase with R-MP, one can modify the Ser240, Thr242, orSer243 residues to accept larger substrates or to favor negativelycharged substrates. For instance, Thr242 can be mutated to Ser to reducethe side chain length. One of the residues can be mutated to lysine orarginine, such as Ser243. The residues (YM-1 numbering) Va130-Va136 arelocated in a beta strand across the active site of theD-aminotransferase, and are also important for activity. Tyr31, Va133,Glu32, and Lys35 are thought to face the active site. Tyr31, Glu32, andVa133 are invariant in all the Bacillus homologs. Ro et al. (FEBS Lett398 (1996) pp. 141-145) mutagenized Va133 to Ala and found a slightlyincreased catalytic efficiency for alpha-ketoglutarate transaminationand a significantly improved catalytic efficiency for bulkier substrates(less steric hindrance). In some homologs Lys35 is replaced with Arg,but if steric hindrance is a concern the Lys residue is preferable.Valines 34 and 36 are also preferable over conservative substitutionssuch as isoleucine, again due to less steric hindrance for largemolecules such as MP.

Example 17 Cloning, Expression, and Testing of Glutamate and AspartateRacemases

This example describes methods used to clone and test amino acidracemase enzymes, which can be used to interconvert between L-glutamateand D-glutamate (or L- and D-aspartate or L- and D-alanine). Glutamate,aspartate, or alanine racemases are useful in a biosynthetic pathway toproduce R,R monatin when a step in that pathway produces an L-amino acid(such as L-glutamate, L-aspartate, or L-alanine) and another step in thepathway consumes a D-amino acid (such as D-glutamate, D-aspartate, orD-alanine). FIG. 4 illustrates a biosynthetic pathway for producing R,Rmonatin from L-tryptophan using an L-tryptophan-specificaminotransferase, an R-specific aldolase, a D-aminotransferase and aglutamate (or aspartate or alanine) racemase.

Genes were cloned into the pET28 and pET30 vectors to generate bothnon-tagged proteins and fusion proteins with cleavable N-terminalHIS₆-Tag/T7-Tags. The resulting proteins were purified using immobilizedmetal affinity chromatography.

Experimental Overview

Genes encoding glutamate racemases (EC 5.1.1.3) from Lactobacillusbrevis (Genbank Accession No. D29627, nucleic acid sequence), andPediococcus pentosaceus (murI gene) (Genbank Accession No. L22789) werecloned and expressed in E. coli. The extracts were tested for activityin conversion of L-glutamate to D-glutamate and D-glutamate toL-glutamate. BioCatalytics aspartate racemase enzyme (EC 5.1.1.13)(BioCatalytics, Pasadena, Calif.) was also tested for interconversionbetween L- and D-aspartate.

Isolation of Genomic DNA for Cloning

L. brevis genomic DNA (ATCC 8287D) was obtained from the American TypeCulture Collection. P. pentosaceus (ATCC 25745) was grown at 37° C. inlactobacilli MRS broth and 2 ml was used for genomic DNA isolation usingthe method of Mekalanos J J. Duplication and amplification of toxingenes in Vibrio cholerae. Cell. 1983. 35:253-63.

Polymerase Chain Reaction Protocol

Primers were designed with 5′ restriction sites and overhangs forcloning into the pET 28 and pET30 vectors (Novagen, Madison, Wis.).

L. brevis glutamate racemase primers: N term: (SEQ ID NO: 401)5′-GCGGCGCCATGGAAAATGATCCGATTGGTCTAATG-3′, and C term: (SEQ ID NO: 402)5′-GCGGCGGTCGACGCAATTACAATTGTGTTTGTC-3′. P. pentosaceus glutamateracemase primers: N term: (SEQ ID NO: 403)5′-GCGGCGCCATGGATGTATGTATAATTTTATTTAG-3′, and C term: (SEQ ID NO: 404)5′-GCGGCGGTCGACAAATTTCATTATTCATTCTAATTT-3′.

The gene derived from L. brevis was amplified using the following PCRprotocol. In a 50 μL reaction 0.150 μg template, 1.6 μM of each primer,0.4 mM each dNTP, 2.8 U Expand High Fidelity™ Polymerase (Roche,Indianapolis, Ind.), 0.5 U Pfu polymerase (Stratagene, La Jolla, Calif.)and 1× Expand™ buffer with Mg were used. The thermocycler program usedincluded a hot start at 96° C. for 3 minutes, 8 repetitions of thefollowing steps: 94° C. for 30 seconds, 52° C. for 45 seconds, and 72°C. for 2 minutes, followed by 22 repetitions of the following steps: 94°C. for 30 seconds, 60° C. for 45 seconds, and 72° C. for 2 minutes.After the 22 repetitions the sample was maintained at 72° C. for 7minutes and then stored at 4° C. This PCR protocol produced a product of˜830 bp, as judged by comparison to DNA size markers.

The gene derived from P. pentosaceus was amplified using the followingPCR protocol. In a 50 μL reaction, 0.15 μg template, 1.6 μM of eachprimer, 0.4 mM each dNTP, 2.8 U Expand High Fidelity™ Polymerase, 0.5 UPfu polymerase and 1× Expand™ buffer with Mg were added. Thethermocycler program used included a hot start at 96° C. for 3 minutes,followed by 8 repetitions of the following steps: 94° C. for 30 seconds,37° C. for 45 seconds, and 72° C. for 2 minutes, followed by 8repetitions of the following steps: 94° C. for 30 seconds, 45° C. for 45seconds, and 72° C. for 2 minutes, followed by 14 repetitions of thefollowing steps: 94° C. for 30 seconds, 55° C. for 45 seconds, and 72°C. for 2 minutes. After the 14 repetitions the sample was maintained at72° C. for 7 minutes and then stored at 4° C. This PCR protocol produceda product of ˜840 bp, as judged by comparison to DNA size markers.

Cloning

The PCR products were gel purified from 0.8% TAE-agarose gels using theQiagen gel extraction kit (Qiagen, Valencia, Calif.). The PCR productswere quantified using a SmartSpec 3000™ spectrophotometer. The productswere digested with restriction enzymes NcoI and SalI following themanufacturer's recommended protocols (New England Biolabs, Beverly,Mass.) and gel purified from 0.8% TAE-agarose gels using the Qiagen gelextraction kit (Qiagen, Valencia, Calif.). Vectors pET28 and pET30 wereprepared by digestion with restriction enzymes NcoI and SalI followed bytreatment with shrimp alkaline phosphatase and purification from 0.8%TAE-agarose gels using the Qiagen gel extraction kit (Qiagen, Valencia,Calif.).

The digested vectors and inserts were ligated using the Rapid™ DNALigation Kit (Roche, Indianapolis, Ind.). Approximately 50 ng of treatedinsert, 100 ng of treated vector (3 to 1 molar ratio of insert tovector), 5 U of T4 DNA ligase, and 1× ligation buffer were incubated for5 minutes at room temperature. The ligation reactions were purifiedusing the High Pure PCR Product Purification Kit (Roche, Indianapolis,Ind.) and used to transform E. coli DH10B electrocompetent cells(Invitrogen, Carlsbad, Calif.). Ten μl of each ligation reaction wasadded to 40 μl of DH10B cells and were transformed by electroporationusing the Bio-Rad Gene Pulser II (Bio-Rad, Hercules, Calif.) under thefollowing conditions: 2.5 kV, 25 μF, 200 ohm in a 0.2 cm cuvette. Thecells were allowed to recover in 1 mL of room temperature SOC for 1 hourat 37° C. with shaking at 225 rpm. Cells were plated on LB platescontaining kanamycin (50 μg/mL).

Plasmid DNA was purified from the resulting transformants using theQiagen spin miniprep kit (Qiagen, Valencia, Calif.) and screened for thecorrect inserts by restriction digest with NcoI and SalI. The sequencesof plasmids appearing to have the correct insert were verified bydideoxy chain termination DNA sequencing.

Gene Expression and Assays

Plasmid DNA, verified by sequence analysis, was subcloned into E. coliexpression host BL21(DE3) (Novagen, Madison, Wis.). The cultures weregrown. The plasmids were isolated using a Qiagen miniprep kit (Qiagen,Valencia, Calif.) and analyzed by restriction digest to confirmidentity.

Induction in BL21(DE3) was initially performed with L. brevis and P.pentosaceus glutamate racemases in both pET28 (untagged) and pET 30(histidine-tagged) vectors. A time course study was performed withcultures grown in 250 mL LB containing kanamycin (50 mg/L) to an OD₆₀₀of 0.5-0.6, induced with 100 mM IPTG (isopropyl thiogalacatoside) andsampled at 0 and 3 hours post induction. Cells from 600 μL (0 hour) and275 μL (3 hour) were resuspended in 40 μL sodium dodecyl sulfate buffercontaining 2-mercaptoethanol, heated at 95° C. for 10 minutes, andcooled. Aliquots of these total cellular protein samples were analyzedby SDS-PAGE using a 4-15% gradient gel.

Cell extracts were also prepared from the 3 hour cultures by suspendingcell pellets from 5 mL of culture in 0.625 mL Novagen BugBuster™ reagent(Novagen, Madison, Wis.) containing 0.625 μL benzonase nuclease and 3 μLprotease inhibitor cocktail set #3 (Calbiochem-Novabiochem Corp., SanDiego, Calif.) at room temperature for 20 minutes with gentle shaking,and centrifuging at 16,000×g to remove cell debris. The supernatants(cell extracts) were loaded onto 4-15% gradient gels for analysis of thecellular soluble proteins.

The 3-hour samples from cloned L. brevis glutamate racemase and P.pentosaceus glutamate racemase showed both total and soluble proteinthat corresponded to the correct size (approximately 31 kDa). The L.brevis pET30 (histidine-tagged) gene product was over-expressed at ahigher level than, and was also more soluble (>20% of soluble protein)than the L. brevis pET 28 (untagged) gene product, as well as the P.pentosaceus gene products in both vectors. The P. pentosaceus geneproduct showed equal overexpression and solubility in the pET28 andpET30 vectors, which was significantly less than that observed for theL. brevis pET30 gene product.

Cells from the induced cultures (250 mL) were centrifuged and washedonce with 0.85% NaCl. Cell pellets were resuspended in 5 mL/g wet cellweight of BugBuster™ (Novagen, Madison, Wis.) reagent containing 5 μL/mLprotease inhibitor cocktail set #3 (Calbiochem-Novabiochem Corp., SanDiego, Calif.) and 1 μL/mL benzonase nuclease. Samples were incubated atroom temperature for 20 minutes on an orbital shaker. Insoluble celldebris was removed by centrifugation at 16,000×g for 20 minutes at 4° C.

Cell extracts were assayed for glutamate racemase activity using thefollowing protocol. 400 μL reactions were carried out in 10 mM potassiumphosphate (pH 8.0), 0.2 mM DTT, and 10 mM L-glutamate or D-glutamate.The reactions were initiated by the addition of 20-100 μL of cell freeextracts and were incubated at room temperature. Sample aliquots weretaken over a time course of 1 minute, 5 minutes, 10 minutes, 20 minutesand 1 hour (zero minute samples served as control reactions). 2M formicacid (25 μL) was added to each 40-μL sample aliquot to stop the reactionand the precipitated protein was removed by centrifugation. Supernatantswere removed and frozen at −80° C. until they were analyzed by LC/MS/MS.

Assay results from cell extracts from pET30 induction with 100 mM IPTG(3 hours) demonstrate that L. brevis (Genbank Accession No. BAA06106.1GI:468450) and P. pentosaceus (Genbank Accession No. AAA16761.1GI:349029) enzymes have significant levels of racemase activity on bothglutamate isomers. The P. pentosaceus racemase (20 μL of cellularextracts) reached equilibrium between L- and D-glutamate in 10-20minutes starting with either substrate. The L. brevis enzyme (20 μL ofcellular extracts) also reached equilibrium in approximately 20 minutes.

A partially purified aspartate racemase enzyme (catalog #ASPR-101)purchased from BioCatalytics, Inc. (Pasadena, Calif.) was assayed foractivity on L-aspartate and D-aspartate using a protocol similar to theone above. The commercial enzyme showed racemase activity on bothisomers. Using 0.5-1 mg of enzyme, equilibrium was achieved in 20-60minutes.

All three racemases (L. brevis glutamate racemase, P. pentosaceusglutamate racemase and BioCatalytics aspartate racemase) were alsoassayed for activity on S,S monatin using the following protocol. 400-μLreactions were carried out in 10 mM potassium phosphate (pH 8.0), 0.2 mMDTT, and 10 mM S,S monatin. The reactions were initiated by the additionof cell free extracts (L. brevis and P. pentosaceus) or purified enzyme(BioCatalytics aspartate racemase) and were incubated at roomtemperature. Sample aliquots were taken over a time course of 1 minute,5 minutes, 10 minutes, 20 minutes and 1 hour (zero minute samples servedas control reactions as well as samples without enzyme). 2M formic acid(25 μL) was added to each 40-μL sample aliquot to stop the reaction andthe precipitated protein was removed by centrifugation. Supernatantswere removed and frozen at −80° C. until they were analyzed by LC/MS/MS(Example 1). No decrease in S,S monatin concentration was noted overtime, nor was there any increase in S,R monatin (present initially as<5% contaminating byproduct, even in the no enzyme control). Therefore,none of the racemases assayed showed activity towards monatin.

Example 18 Production of R,R Monatin from L-tryptophan Using Glutamateor Aspartate Racemases

This example describes methods of producing stereoisomerically-enrichedR,R monatin from L-tryptophan using an L-tryptophan (L-tyrosine, oraromatic) aminotransferase, ProA aldolase, glutamate or aspartateracemase, and a broad specificity D-amino acid aminotransferase. FIG. 5is a diagram that illustrates the pathway. This approach to productionof stereoisomerically enriched R,R monatin requires an enzyme for step 1that has low activity in the production of monatin from monatinprecursor (MP). Based upon earlier results, we used the Sinorhizobiummeliloti and Rhodobacter sphaeroides tatA gene products described inExample 1 from WO 03/091396 A2.

Materials and Methods

Glutamate racemases from L. brevis and P. pentosaceus were produced inE. coli as described in Example 17. In some cases the His₆-taggedversion of these enzymes were purified using His-Bind 900 cartridgesaccording to manufacturer's protocols (Novagen, Madison, Wis.) and weredesalted to remove imidazole using PD-10 columns (G25 Sephadex, GEHealthcare, Piscataway, N.J.). The enzymes were eluted in 25 mMpotassium phosphate pH 8.0. Aspartate racemase (ASPR-101) andD-aminotransferase (AT-103) were purchased from BioCatalytics, Inc.(Pasadena, Calif.). S. meliloti and R. sphaeroides tyrosine (aromatic)aminotransferases were prepared as described in Example 1 from WO03/091396 A2. Comamonas testosteroni ProA aldolase was prepared asdescribed in Example 4 from WO 03/091396 A2. Total protein assays weredone utilizing the Bio-Rad Protein Assay according to manufacturer'sprotocols (Hercules, Calif.).

Reduction in Amount of S,S Monatin Produced Using Racemases

Reaction mixtures (1 mL volume, run in duplicate) contained 100 mMpotassium phosphate buffer (pH 8), 2 mM MgCl₂, 0.05 mM pyridoxal5′-phosphate (PLP), 200 mM sodium pyruvate, 5 mM sodium α-ketoglutarateor oxaloacetate, approximately 280 μg/mL S. meliloti TatA supplied in acellular extract, 1 mg/mL BioCatalytics D-aminotransferase (AT-103)(BioCatalytics, Pasadena, Calif.), 100 μL/mL of glutamate racemasecellular extract or 1 mg/mL aspartate racemase, and approximately 100μg/mL of ProA aldolase provided as a cellular extract. Solid tryptophanwas added at a concentration of 10.2 mg/ml. Negative controls did notcontain racemase. Samples were incubated at 30° C. (shaking at 250 rpm)for 1 hour, 2 hours, or overnight. Samples were centrifuged to removeprecipitate, syringe filtered, and stored at −80° C. prior to analysisfor monatin using the LC/MS/MS method described in Example 1. Most ofthe samples contained >95% S,S monatin, due to the amounts of nativeL-aminotransferase present in the cellular extracts. However, thesamples that contained racemase had a reduced amount of total monatin asa result of the racemase enzymes making L-glutamate less available fortransamination of MP. Without racemase, 1545-2355 ppm monatin(predominantly S,S) was produced during the timecourse. With theracemases present, only 340-879 ppm (L. brevis enzyme), 444-531 ppm (P.pentosaceus enzyme), and 506-1460 ppm monatin (aspartate racemase) wereproduced. These data indicate that the racemases are active in thereaction conditions required to produce monatin. To minimize formationof S,S monatin from cellular extract enzymes such as aspartateaminotransferases, further experiments were done with purified enzymesand a higher ratio of D-aminotransferase to L-aminotransferase enzymes.

Conversion of L-Tryptophan to 4-R Containing Isomers of Monatin

The above experiments were repeated using approximately 54 μg ofpurified L-aminotransferase (either S. meliloti or R. sphaeroides TatA),1 mg aspartate aminotransferase (BioCatalytics, Pasadena, Calif.), 1 mgD-aminotransferase, oxaloacetate as the amino acceptor, and 75 μgpurified aldolase. Reactions were run in duplicate with a 2-hoursampling time and an overnight incubation time. Negative controls weredone with S. meliloti L-aminotransferase but no racemase. In addition toquantification of R,R/S,S and S,R/R,S monatin peak quantification basedon reversed phase chromatography, the percentage of each stereoisomerwas determined using the FDAA derivitization technique described inExample 1. The results are as follows:

TABLE 22 Total Incubation Monatin % % % % L-Aminotransferase Time (ppm)S,S R,R R,S S,R S. meliloti TatA 2 hr 17.1 10.2 58.1 0.8 31.0 S.meliloti TatA 2 hr 15.8 13.3 55.3 1.0 30.4 S. meliloti TatA overnight77.7 25.8 40.0 1.3 32.9 S. meliloti TatA overnight 67.9 29.4 37.3 1.531.8 R. sphaeroides TatA 2 hr 241.2 96.3 2.3 0.8 0.6 R. sphaeroides TatA2 hr 223.2 95.7 2.7 1.0 0.6 R. sphaeroides TatA overnight 600.4 96.6 1.80.5 1.1 R. sphaeroides TatA overnight 618.5 96.1 2.1 0.5 1.3 no racemasecontrol 2 hr 7.1 92.0 1.4 6.6 0.0 no racemase control 2 hr 5.7 94.0 1.24.8 0.0 no racemase control overnight 44.6 93.5 1.3 4.7 0.5 no racemasecontrol overnight 37.5 95.4 0.9 3.7 0.0

Clearly the presence of the racemase increased the total amount ofmonatin produced when S. meliloti TatA was used as the enzyme forL-tryptophan transamination. Monatin levels increased from an average of6.4 to 16.5 ppm in the two-hour assay, and from 41-73 ppm in theovernight assay. Additionally, the percent of R,R formed increased fromabout 1% up to as much as 58% by utilizing the racemase enzyme. The S,Rstereoisomer of monatin, another potent sweetener, was the other majorcomponent, increasing from nearly 0 in the negative controls to 31%. TheR. sphaeroides TatA clearly had more activity on S-MP then the S.meliloti L-transaminase, demonstrating the importance of having anenzyme that has a high substrate specificity for L-tryptophan ascompared to MP when 4-R isomers of monatin are the desired products.With about 10% of the total monatin being 4S at the two-hour time point,the S. meliloti TatA could be considered as having limited activity onMP.

The experiments were repeated with the purified S. meliloti TatA (54 μg)and the L. brevis glutamate racemase. When purified glutamate racemasewas used, approximately 64 μg was used per 1 mL reaction. Cellularextracts containing the glutamate racemase were also tested, and 1.4 mgof soluble protein was used. A no racemase negative control was utilizedagain, and all samples were run in duplicate. The results are asfollows:

TABLE 23 Total Incubation Monatin % % % % Glutamate racemase Time (ppm)S,S R,R R,S S,R L. brevis (purified) 2 hr 3.3 49.1 34.2 3.7 13.0 L.brevis (purified) 2 hr 3.6 47.9 35.2 3.5 13.4 L. brevis (purified)overnight 29.3 58.9 24.7 3.2 13.2 L. brevis (purified) overnight 40.255.1 25.0 4.7 15.3 L. brevis (cell extract) 2 hr 10.5 45.8 35.9 1.1 17.2L. brevis (cell extract) 2 hr 10.5 47.4 33.9 1.1 17.6 L. brevis (cellextract) overnight 79.4 70.3 17.9 1.3 10.5 L. brevis (cell extract)overnight 80.1 69.1 19.1 1.1 10.7 none 2 hr 2.7 84.1 7.1 6.3 2.4 none 2hr 3.2 84.9 6.0 6.8 2.2 none overnight 36.5 92.3 2.3 4.2 1.2 noneovernight 30.5 92.7 2.0 4.1 1.3

Again, it is clear that the addition of the racemase increases the totalmonatin produced from L-tryptophan, as well as increasing the relativeamounts of 4R-containing isomers of monatin as compared to S,S monatin.The use of purified aldolase, racemase, and L-aminotransferase greatlyimproves the ability to control the desired stereoisomer formation. Theratio of L to D aminotransferase is also a way to manipulatestereochemistry of the final product.

When comparing results shown in Tables 18 and 19 in Example 13 toresults with reaction conditions similar to the conditions above, onecan see that approximately 7-29 ppm of monatin were formed fromindole-3-pyruvate and the percentages of R,R monatin formed wereapproximately 51-90%. Using the aspartate racemase increased the totalamount of monatin produced to 16-78 ppm monatin with % R,R ofapproximately 40-58%. Additionally, a more stable and less expensive rawmaterial, L-tryptophan, was utilized. In Example 14, approximately 73ppm monatin was produced from D-tryptophan at a ratio of R,R:S,R ofapproximately 1.7:1. The total amount of 4R isomers was >80% of thetotal monatin. Because both R,R-monatin and S,R-monatin are potentsweeteners (>1000 times sweeter than sucrose) the ability to enrich forthese isomers without the need for expensive D-amino acid substrates iscritical.

As described in Examples 13 and 14, D-alanine can serve as the aminodonor for transamination of MP to monatin. Many L-aminotransferases havethe ability to utilize pyruvate as an amino acceptor to some extent, andproduce L-alanine. Because the above-mentioned reactions use highconcentrations of pyruvate, it is likely that some of the pyruvate isconverted to L-alanine. For example, during transamination ofL-tryptophan, the HexAspC enzyme described in Example 16 has been foundto convert 10-18% of pyruvate (50-200 mM initial concentrations) toL-alanine in 2 hours if alpha-ketoglutarate is absent. The enzyme showeda 10-fold preference for alpha-ketoglutarate when both amino acceptorswere present at high (>50 mM) concentrations. AspC (described in WO03/091396 A2) also produced some L-alanine from pyruvate. Therefore, itis expected that one can omit the addition of alpha-ketoglutarate oroxaloacetate in the above reactions, and utilize an alanine racemase (EC5.1.1.1) in place of glutamate or aspartate racemase. Alanine racemaseenzymes were first identified in Brucella abortus and Streptococcusfaecalis (Marr, A. G. and Wilson, P. W. Arch. Biochem. Biophys., 49(1954) 424-433 and Wood, W. A. and Gunsalus, I. C. J. Biol. Chem., 190(1951) 403-416). The dadB gene in Salmonella typhimurium was identifiedas the source of alanine racemase activity, and several hundred homologscan be found in genomics databases. Other known sources of alanineracemase activity are Escherichia coli, Bacillus subtilis, Pseudomonasaeruginosa, Vibrio cholerae, Schizosaccaroyces pombe, and Bacilluscereus. A basidiomycetous mushroom, Lentinus edodes, also contains abroad activity alanine racemase. A thermostable homolog from Bacillusstearothermophilus is available for purchase from Sigma-Aldrich (catalog#A8936) (Sigma-Aldrich, St. Louis, Mo.) and has been immobilized forcommercial applications (Inagaki, K., Biochemistry, 25: 3268 1986). Analanine racemase is converted to a glutamate or aspartate racemase byrandom methods such as mutagenic PCR, passage through mutagenic strains,or other methods to those known in the art. A more focused evolution ofthe alanine racemase is focused on active site residues, including Lys129, Met134, and the residues including and between Gly283 and Trp288(numbering from Bacillus stearothermophilus).

Example 19 D-phenylglycine aminotransferase (D-4-hydroxyphenylglycineaminotransferase)

As shown in FIG. 3, D-phenylglycine aminotransferase is useful in abiosynthetic pathway for the production of monatin. For example,D-phenylglycine aminotransferase produces R,R monatin from R-MP withL-glutamate as the amino donor.

PCR synthesis of P. stutzeri 4 D-hydroxyphenylglycine aminotransferasefrom oligonucleotide primers

This example describes methods that were used to synthesize 4D-hydroxyphenylglycine aminotransferase, a stereoinverting enzyme thatcan be used to convert R monatin precursor to R,R monatin usingL-glutamate as the amino donor.

Primer Design

The published sequence (Genbank Accession No. AY319935, nucleic acidsequence; Genbank Accession No. AAQ8290, protein sequence) forPseudomonas stutzeri 4 D-hydroxyphenylglycine aminotransferase (4 D-HPGAT) was used as a template for PCR primer design. Alternatively, the4-D-hydroxyphenylglycine aminotransferase from Pseudomonas putida,(CAD42450 (protein), AX467211 (nucleotide)) is used as a sequencetemplate. A total of 34 forward primers and 35 reverse primers weredesigned; forward and reverse primers were 40-mers sharing 20overlapping base pairs. In addition, 2 outer primers were designed with5′ restriction sites and overhangs for cloning into the pET 28 and pET30vectors (Novagen, Madison, Wis.).

P. stutzeri 4 D-HPG AT outer primers: N term (with NdeI Site): (SEQ IDNO: 405) 5′-GGCCGGCATATGTCGATCCTTAACGACTACAAACGT-3′, and C term (withXhoI site): (SEQ ID NO: 406) 5′-GGAAGGCTCGAGTCATGATTGGTTTCCAGACAAATT-3′.

Polymerase Chain Reaction Protocol

The gene sequence from P. stutzeri was amplified using the followingprotocols. The primary 100 μL PCR reaction included 0.05 μM of each ofthe internal 69 primers, 0.4 mM each dNTP, 10 U rTth Polymerase XL(Roche, Indianapolis, Ind.), 0.625 U Pfu polymerase (Stratagene, LaJolla, Calif.), 1×XL buffer and 1 mM Mg(OAc)₂. The thermocycler programused included a hot start at 94° C. for 3 minutes, 15 repetitions of thefollowing steps: 94° C. for 30 seconds, 42° C. for 30 seconds, and 68°C. for 15 seconds, followed by 10 repetitions of the following steps:94° C. for 30 seconds, 52° C. for 30 seconds, and 68° C. for 30 seconds,followed by 10 repetitions of the following steps: 94° C. for 30seconds, 60° C. for 30 seconds, and 68° C. for 1 minute and 15 seconds.After the final 10 cycles the sample was maintained at 68° C. for 7minutes and then stored at 4° C. This PCR protocol produced a smear ofproduct at −0.5 kb on a 0.8% TAE-agarose gel.

The secondary PCR reaction was set up using the primary PCR reaction astemplate. The secondary 100 μL PCR reaction included 2.5 μL of theprimary PCR reaction, 0.5 μM of each of the 2 outer primers (with NdeIand XhoI restriction sites), 0.4 mM each dNTP, 10 U rTth Polymerase XL,0.625 U Pfu polymerase, 1×XL buffer and 1 mM Mg(OAc)₂. The thermocyclerprogram used included a hot start at 94° C. for 3 minutes, 10repetitions of the following steps: 94° C. for 30 seconds, 52° C. for 30seconds, and 68° C. for 1 minute 30 seconds, followed by 15 repetitionsof the following steps: 94° C. for 30 seconds, 60° C. for 30 seconds,and 68° C. for 1 minute 30 seconds. After the 15 repetitions, the samplewas maintained at 68° C. for 7 minutes and then stored at 4° C. This PCRprotocol produced a distinctive product band at ˜1.4 kb on a 0.8%TAE-agarose gel.

The PCR product was gel purified from 0.8% TAE-agarose gel using theQiagen gel extraction kit (Qiagen, Valencia, Calif.). The product wasTOPO cloned and transformed into TOP 10 cells according tomanufacturer's protocol (Invitrogen, Carlsbad, Calif.). Plasmid DNA waspurified from the resulting transformants using the Qiagen spin miniprepkit (Qiagen, Valencia, Calif.) and screened for the correct inserts byrestriction digest with NdeI and XhoI. The sequences of plasmidsappearing to have the correct insert were verified by dideoxy chaintermination DNA sequencing with universal M13 forward and M13 Reverseprimers. Of the 10 clones sequenced, all had at least one mutation fromthe desired sequence. The best clone had a single base-pair mutationthat resulted in an amino acid change. The sequence of this clone wascorrected using the QuickChange Mutagenesis protocol according tomanufacturer recommendations (Stratagene, La Jolla, Calif.).

The corrected TOPO clone was digested with restriction enzymes NdeI andXhoI following the manufacturer's recommended protocols (New EnglandBiolabs, Beverly, Mass.) and gel purified from 0.8% TAE-agarose gelsusing the Qiagen gel extraction kit (Qiagen, Valencia, Calif.). VectorspET 28 and pET 30 were prepared by digestion with restriction enzymesNde1 and Xho1 followed by treatment with shrimp alkaline phosphatase andpurification from 0.8% TAE-agarose gels using the Qiagen gel extractionkit (Qiagen, Valencia, Calif.).

The digested vectors and insert were ligated using the NEB QuickLigation Kit (Beverly, Mass.). Approximately 50 ng of treated insert,100 ng of treated vector (3 to 1 molar ratio of insert to vector), 5 Uof T4 DNA ligase, and 1× ligation buffer were incubated for 5 minutes atroom temperature. The ligation mixture was transformed in to TOP10F′chemically competent cells (Invitrogen, Carlsbad, Calif.). The cellswere allowed to recover in 0.25 mL of room temperature SOC for 1 hour at37° C. with shaking at 225 rpm. Cells were plated on LB platescontaining kanamycin (50 μg/mL). Plasmid DNA is purified from theresulting transformants using the Qiagen spin miniprep kit (Qiagen,Valencia, Calif.) and screened for the correct inserts by restrictiondigest with NdeI and XhoI.

Gene Expression and Assays

Plasmid DNA was transformed into E. coli expression host BL21(DE3)(Novagen, Madison, Wis.). The cultures were grown and the plasmids wereisolated using Qiagen miniprep kit (Qiagen, Valencia, Calif.), andanalyzed by restriction digest to confirm identity.

Induction in BL21(DE3) is initially performed with P. stutzeri 4 D-HPGin both pET 28 (histidine-tagged) and pET 30 (untagged) vectors. A timecourse study is performed with cultures grown in 250 mL LB containingkanamycin (50 mg/L) to an OD₆₀₀ of 0.5-0.6, induced with 100 mM IPTG(isopropyl thiogalacatoside) and sampled at 0 and 3 hours postinduction. An appropriate volume of cells from 0 hours and 3 hours isresuspended in 40 μL sodium dodecyl sulfate buffer containing2-mercaptoethanol and heated at 95° C. for 10 minutes, and cooled.Aliquots of these total cellular protein samples is analyzed by SDS-PAGEusing a 4-15% gradient gel.

Cell extracts are also prepared from the 3 hour cultures by suspendingcell pellets from 5 mL of culture in 0.625 mL Novagen BugBuster™ reagent(Novagen, Madison, Wis.) containing 0.625 μL benzonase nuclease and 3 μLprotease inhibitor cocktail set #3 (Calbiochem-Novabiochem Corp., SanDiego, Calif.) at room temperature for 20 minutes with gentle shaking,and centrifuging at 16,000×g to remove cell debris. The supernatants(cell extracts) are loaded onto 4-15% gradient gels for analysis of thecellular soluble proteins. When required, protein is purified usingHis-Bind 900 cartridges according to manufacturer's protocols (Novagen,Madison, Wis.) and is desalted to remove imidazole using PD-10 columns(G25 Sephadex, GE Healthcare, Piscataway, N.J.).

Organisms with D-phenylglycine aminotransferase (DPGAT)

Organisms of the genus Pseudomonas and like genera, with astereoinverting D-phenylglycine aminotransferase (also calledD-4-hydroxyphenylglycine aminotransferase) are isolated in the followingmanner. Soil samples are incubated on petri plates with the followingmedium: (per liter) 15 g Agar, 3.4 g KH₂PO₄, 3.55 g Na₂HPO₄, 0.2 gMgSO₄.7H₂O, 8 mg CaCl₂.2H₂O, 10 mg yeast extract, 1 ml 1000× traceelements solution, 1 g D-phenylglycine (D-4-hydroxyphenylglycine).

Isolates are tested by PCR for the presence of a stereoinvertingaminotransferase (primers designed from known D-phenylglycineaminotransferases) or are further enriched for the presence of astereoinverting aminotransferase as follows: isolated from the platescould be grown in liquid medium as above minus the agar at 30° C. withshaking to an OD₆₀₀ of about 1.0. Cells are harvested by centrifugationand washed twice with 0.85% NaCl. A 10 mg (wet weight) sample issuspended in 1 ml potassium phosphate buffer (pH 7.0) and 5 mMD-phenylglycine (or D-4-hydroxyphenylglycine). Neutralized 15 mM(aminooxy)acetic acid is added to duplicate samples prepared asdescribed above. Consumption of D-phenylglycine (or D-4-hydroxyglycine)is measured by HPLC. Isolates capable of degrading D-phenylyglycine (orD-4-hydroxyphenylglycine), but do so at a slower rate in the presence of(aminooxy)acetic acid are selected for further analysis. Isolates aretested, by PCR, for the presence of a stereoinverting aminotransferase(primers designed from known D-phenylglycine aminotransferases).

The presence of the stereoinverting aminotransferase is confirmed bygrowing a culture in liquid medium as described above, harvesting thecells and making a cell free crude extract (CFE) and testing forD-phenylglycine aminotransferase (or D-4-hydroxyphenylglycineaminotransferase) enzyme activity. CFE is added to a reaction mixturewith the following final concentrations: 0.1M CAPS (pH 9.5), 60 mML-glutamate (sodium salt), 5 mM benzoylformate (or 4-hydroxybenzoate)and 50 μM PLP. The reverse reaction is measured by adding CFE to areaction mixture with the following concentrations: 50 mM potassiumphosphate (pH 7.0), 60 mM D-phenylglycine (or D-4-hydroxyphenylglycine),5 mM α-ketoglutarate, 50 μM PLP. The assays are incubated at 35° C. andaliquots are taken at time points and stopped by boiling for 2 minutes.Product will be quantitated by the HLPC method of Gil-Av, E., Tishbee,A., Hare, P. E., Resolution of underivatized amino acids by reversedphase chromatography. J. Am. Chem. Soc., 102: 5115-5117 (1980) or bymethods described in Example 1 (measurement of glutamate formation).

As an alternative to PCR based methods, the stereoinvertingD-phenylglycine aminotransferase is purified from the isolated bacteriaby conventional protein purification techniques including ammoniumsulfate fractionation, and conventional column chromatography. Once theprotein has been purified to a reasonable degree peptide microsequencingtechniques or conventional Edman type amino acid sequencing are utilized(see golgi.harvard.edu/microchem/for descriptions of the protocols andequipment used for this type of work). Degenerate primers are designedbased on sequence available from the closest known relative of theprotein source. Degenerate PCR and genome walking is then performedaccording to established protocols to isolate the stereoinvertingD-phenylglycine aminotransferase coding sequence.

DPGAT Monatin Production

D-hydroxyphenylglycine aminotransferases, as described in (1) and (2)above, are used in crude cell free protein extracts or purified asdescribed in (1) above. S. meliloti and R. sphaeroides tyrosine(aromatic) aminotransferases are prepared as described in Example 1 fromWO 03/091396 A2. Comamonas testosteroni ProA aldolase is prepared asdescribed in Example 4 from WO 03/091396 A2. Total protein assays aredone utilizing the Bio-Rad Protein Assay according to manufacturer'sprotocols (Hercules, Calif.).

Reaction mixtures (1 mL volume, run in duplicate) contain 100 mMpotassium phosphate buffer (pH 8), 2 mM MgCl₂, 0.05 mM pyridoxal5′-phosphate (PLP), 200 mM sodium pyruvate, 5 mM sodium α-ketoglutarate,approximately 280 μg/mL S. meliloti TatA supplied in a cellular extract,100 μL/mL of D-hydroxyphenylglycine aminotransferase cellular extract or1 mg/mL purified D-hydroxyphenylglycine aminotransferase, andapproximately 100 μg/mL of ProA aldolase provided as a cellular extract.Solid tryptophan is added at a concentration of 10.2 mg/ml. Negativecontrols are set up without D-hydroxyphenylglycine aminotransferase.Samples are incubated at 30° C. with gentle shaking for ˜1 hour orovernight. Samples are centrifuged to remove precipitate, syringefiltered, and stored at −80° C. prior to analysis for monatin using theLC/MS/MS method described in Example 1.

D-hydroxyphenylglycine aminotransferases with improved activity formonatin production are made my mutagenesis techniques known to those inthe art, including: mutagenic PCR, passage through mutagenic strains, orsite-directed mutagenesis. The improved D-hydroxyphenylglycineaminotransferases are selected by growth on minimal medium withR,R-monatin as the source of nitrogen. Initially the selection is basedon growth but as improved aminotransferases are selected the screen isgrowth rate based. That is, cells with mutated versions of the gene aregrown and the gene is expressed in minimal medium with R,R-monatin asthe nitrogen source. The growth rates of the cells with the mutatedversions of the gene are compared to the unmutated version. Those cellswith a faster growth rate are selected and the aminotransferase isanalyzed further. The D-hydroxyphenylglycine aminotransferase ismutagenized by available techniques such as error-prone PCR and passagethrough mutagenic strains or by methods for which a license has beenobtained such as DNA shuffling and other directed evolutiontechnologies.

DPGAT assay

Cells with the recombinant D-hydroxyphenylglycine aminotransferase weregrown, the protein expressed, and the cells lysed as described inExample 17 or by standard protocols. The protein is expressed in itsnative host using described methods (Wiyakrutta, W., Meevootisom, V. Astereoinverting D-phenylglycine aminotransferase from Pseudomonasstutzeri ST-201: purification, characterization, and application forD-phenylglycine synthesis. J. Biotechnol., 55: 193-203 (1997)).

Cell free extract was added to a reaction mixture with the followingfinal concentrations: 100 mM potassium phosphate (pH 7.0-8.5), 60 mMD-phenylglycine (or D-4-hydroxyphenylglycine), 5 mM α-ketoglutarate, 50μM PLP. The assays were incubated at room temperature and aliquots weretaken at time points and stopped by adding an equal volume of formicacid. Product (L-glutamate) is quantitated by methods described inExample 1.

Example 20 Discovery of a D-Methionine Aminotransferase Gene BACKGROUND

D-methionine-pyruvate aminotransferase (EC 2.6.1.41) is thought to beanother example, although rare, of a stereoinverting transaminase. Thisenzyme catalyzes the reversible conversion of D-methionine and pyruvateto L-alanine and 4-methylthio-2-oxobutanoate. Oxaloacetate,phenylpyruvate, 2-oxobutyrate, 2-oxovalerate, 2-oxoheptanoate,glyoxylate, and oxoglutarate can also serve as amino acceptors.

Transamination of D or L methionine is thought to be part of a pathwayto ethylene production in higher plants (cauliflower, tomato, apple, peastem, banana, peanut) as well as in soil microorganisms (Escherichiacoli, Pseudomonas pisi, Pseudomonas aeruginosa, Bacillus mycoides,Acinetobacter calcoaceticus, Aeromonas hydrophila B12E, Rhizobiumtrifolii N2P7, Penicillium digitatum, Saccharomyces cerevisiae,Corynebacterium D7F). D. C. Billington, B. T. Golding, and S. B.Primrose Biochem J. (1979) 182, 827-836. In bacteria, L-methionine istypically used as the substrate in the ethylene production studies andbroad specificity enzymes such as TyrB or AspC from E. coli are thoughtto be responsible for the transamination. However, S. B. Primrose J.Gen. Microbiol. (1976), 95(1), 159-65 and S. B. Primrose J. Gen.Microbiol. (1977), 98, 519-528. showed that E. coli strain SPA O(University of Warwick culture collection) produced nearly as muchethylene from D-methionine as from L-methionine in batch cultures.Because no broad specificity D-aminotransferase has been identified inE. coli, one possible explanation could be that the E. coli D-amino aciddehydrogenase (encoded by the dadA gene) converts the D-methionine to4-methylthio-2-oxobutanoate. It is also possible that there is amethionine racemase in E. coli; however, no such enzyme has beendescribed in the literature.

In contrast to E. coli, in cauliflower florets (mitochondrial extractpreparations) and germinating peanut seeds production of ethylene washigher when D-methionine and pyruvate were supplied to the enzymeextract as compared to L-methionine and pyruvate (L. W. Mapson, J. F.March, and D. A. Wardale Biochem J. (1969) 115, 653-661; J. I. Durham,P. W. Morgan, J. M. Prescott and C. M. Lyman Phytochemistry (1973) 12,2123-2126). Therefore the possibility of a combination of methionineracemase and an L-aminotransferase is not supported by the data.Dehydrogenase activity was ruled out by dialysis of cellular extracts ofcauliflower, no NAD was present in the assay mixtures. Oxidase activitywas ruled out as no consumption of oxygen was noted and there was norequirement for FAD. The D-methionine aminotransferase from peanuttissues was purified, shown to be dependent on PLP, and shown to beindependent of L-methionine aminotransferase activity. There is apossibility that these D-methionine—pyruvate aminotransferases actuallyproduce D-alanine as a byproduct (similar to the Bacillus enzymesdescribed in Examples 13 and 14), and that the cells contain alanineracemase to recycle the D-alanine back to L-alanine (or an analogousamino donor). In either case, discovery of the broad specificityD-aminotransferase from higher plants is advantageous for development ofprocesses that produce R,R monatin or S,R monatin.

Experimental Overview

D-methionine aminotransferase is partially purified from cauliflowerflorets and germinating peanut embryos using standard chromatographyprotocols and a Pharmacia AKTA Explorer system. The protein sequences ofhomologous proteins are determined by LC/MS/MS fingerprinting techniquesand database searching performed by Harvard Microchemistry facility. Thecoding regions of the plant genes are cloned from a cDNA library usingstandard PCR protocols or by synthesis of the gene as described inExample 19.

Alternatively, cDNA expression libraries are constructed (Stratagene, LaJolla, Calif.) from cauliflower tissue or peanut seeds grown in thepresence of D-methionine (and producing ethylene). The libraries aretransformed into E. coli methionine auxotrophs from the E. coli GeneticStock Center (Yale) such as strains RC519 or AB1931. Plasmids of strainscapable of growth on minimal media containing D-methionine contain thecoding region of interest (see Example 15, an analogous screeningtechnique).

Once the coding regions of interest are obtained and are expressed in astandard E. coli laboratory strain, the resulting gene products can beused in assays to produce R,R monatin as described in Example 19 inplace of the D-hydroxyphenylglycine aminotransferase, with the exceptionof the pH being 7.5 (the optimal pH for the aminotransferase). If theD-methionine aminotransferase has a strict requirement for D-amino aciddonor substrates, the enzyme can be used to make R,R monatin asdescribed in Examples 13 and 14. The gene can be mutagenized andscreened for increased activity as described in Example 19.

Methods

Isolation from Cauliflower

Four hundred grams of freshly picked cauliflower florets are extractedwith 400 mL of a 4° C. sucrose/buffer solution (0.4M sucrose and 0.1Msodium phosphate buffer pH 7.4) by alternating soaking and mixing usinga blender. Cell debris is removed by filtration with cheesecloth and theresulting solution is centrifuged at 40,000×g for 30 minutes at 4° C.The solid material (containing mitochondrial organelles) is resuspendedin 20 mL 10 mM sodium phosphate buffer pH 7.4, and enzymes are extractedwith 200 mL cold (−30° C.) acetone. The suspension is recentrifuged andthe precipitate is dried using a Savant Speed Vac. The solid material isdissolved in 10 mM sodium phosphate buffer pH 7.4, and residual acetoneis removed using a PD-10 column (GE Healthcare, Piscataway, N.J.).

Aminotransferase activity is assayed by incubation of the enzymepreparation with 5 mM D-methionine, 1 mM pyruvate, 0.05 mM PLP and 2 mMEDTA in 0.1M sodium phosphate buffer pH 7.4. Assays are performed at 25°C. for 16 hours. The 4-Methylthio-2-oxobutanoate is measured byformation of the 2,4-dinitrophenylhydrazone derivative, using LC/MS (m/zof 328) and similar methodology described in Example 1. A 0.4% (w/v)solution of 2,4-dinitrophenylhydrazine in 2M sulfuric acid is prepared,and a half volume is added to the assay mixture after incubation. Themixture is mixed with gentle shaking at 30° C. for 30 minutes and theprecipitate is collected by centrifugation and analyzed by LC/MS.Protein fractions separated by standard chromatographic techniques areassayed for activity in a similar manner, but the co-product alanine ismeasured by the OPA post-column derivitization technique described inExample 1.

Isolation from Peanut (Arachia hypogea L. cv. Stan)

The D-methionine aminotransferase enzyme from germinating peanut embryohomogenate (minus the cotyledons) is purified according to the method ofJ. I. Durham, P. W. Morgan, J. M. Prescott and C. M. LymanPhytochemistry (1973) 12, 2123-2126. Reducing agents are used during thepreparation of crude extracts to stabilize the enzymes, and the celldebris is removed by centrifugation at 33,000×g. A 35-50% ammoniumsulfate fraction is further purified by incubation at low temperature,and by removal of the proteins in the precipitate. The supernatant isfurther fractionated using acetone. The active pools are then furtherpurified by gel filtration chromatography (Sephadex 200, GE Healthcare,Piscataway, N.J.).

As protein fractions become enriched with the transaminase protein,2D-gel electrophoresis is utilized to separate the enzyme of interestfor microsequencing. After elucidation of homologous coding regions inplant sequences deposited at NCBI, the D-aminotransferase protein isproduced recombinantly in Escherichia coli using standard molecularbiology techniques. It is expected that the cellular extracts fromcauliflower florets or peanut seeds or recombinantly produced homologousenzymes can be used in production of R,R monatin as described in Example19 (if a stereoinverting transaminase) or Examples 13 and 14 (if a broadspecificity D-aminotransferase).

Example 21 L-alanine aminotransferase/alanine racemase/D-alanineaminotransferase

FIGS. 4, 6, and 8 illustrate biosynthetic pathways for producingstereoisomerically-enriched R,R monatin from L-tryptophan using aL-amino acid aminotransferase (such as L-alanine-aminotransferase and/orL-tryptophan-aminotransferase), an R-specific aldolase, an alanineracemase and a D-alanine aminotransferase.

A tryptophan-specific aminotransferase is described in Example 16.Alternatively, S. meliloti and R. sphaeroides tyrosine (aromatic)aminotransferases are prepared as described in Example 1 from WO03/091396 A2. Comamonas testosteroni ProA aldolase is prepared asdescribed in Example 4 from WO 03/091396 A2. Total protein assays aredone utilizing the Bio-Rad Protein Assay according to manufacturer'sprotocols (Bio-Rad, Hercules, Calif.). Alanine racemase is purchasedfrom Sigma-Aldrich (St. Louis, Mo.) (catalog number A8936). D-alanineaminotransferase is purchased from BioCatalytics (catalog number AT-103)(BioCatalytics, Pasadena, Calif.).

L-alanine aminotransferases are widely distributed in eukaryotes,bacteria, and archaea. The following organisms have been identified(based on sequence homology) as containing an L-alanine aminotransferase(E.C. 2.6.1.2): Arabidopsis thaliana, Ashbya gossypii, Azotobactervinelandii, Bifidobacterium longum, Caenorhabditis elegans, Candidaalbicans, Candida glabrata, Chlamydomonas reinhardtii, Cryptococcusneoformans, Debaryomyces hansenii, Homo sapiens, Hordeum vulgare,Kluyveromyces lactis, Magnaporthe grisea, Medicago truncatula, Musmusculus, Neurospora crassa, Oryza sativa, Phanerochaete chrysosporium,Pinus taeda, Pseudomonas putida, Pyrococcus abyssi, Pyrococcus furiosus,Pyrococcus horikoshii, Rattus norvegicus, Saccharomyces cerevisiae,Schizosaccharomyces pombe, Takifugu rubripes, Trypanosoma cruzi, Vibriocholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Yarrowialipolytica, and Zea mays. Additionally many aminotransferases havelow-level alanine aminotransferase activity and given high levels ofL-glutamate and pyruvate can convert it to L-alanine andα-ketoglutarate. An enzyme with low activity is improved with standardmutagenesis techniques such as error-prone PCR and passage throughmutagenic strains or by directed evolution techniques. The gene for anL-alanine aminotransferase is cloned using publicly available to designprimers and using standard techniques to amplify, clone, express andpurify the gene/enzyme.

Reaction mixtures (1 mL volume, run in duplicate) contain 100 mMpotassium phosphate buffer (pH 8), 2 mM MgCl₂, 0.05 mM pyridoxal5′-phosphate (PLP), 200 mM sodium pyruvate, 5 mM sodium α-ketoglutarate,approximately 280 μg/mL S. meliloti TatA supplied in a cellular extract(or other L-tryptophan specific aminotransferase), 100 μL/mL of alanineracemase cellular extract or 1 mg/mL purified alanine racemase,approximately 280 μg/mL D-alanine aminotransferase supplied in acellular extract and approximately 100 μg/mL of ProA aldolase providedas a cellular extract. Solid tryptophan is added at a concentration of10.2 mg/ml. Negative controls are set up without alanine racemase.Samples are incubated at 30° C. with gentle shaking for ˜1 hour orovernight. Samples are centrifuged to remove precipitate, syringefiltered, and stored at −80° C. prior to analysis for monatin using theLC/MS/MS method described in Example 1. In these reaction mixtures, ifthe L-tryptophan aminotransferase can use α-ketoglutarate, but notpyruvate, as an amino acceptor, one can add an L-alanineaminotransferase, which converts L-glutamate and pyruvate and toL-alanine and α-ketoglutarate, as shown in FIG. 6.

Example 22 Subcloning of Genes Encoding the Aldolases of SEQ ID NO: 276,244, 218, 228, 162, 50, 74, 44, and 116

The genes encoding aldolases having the amino acid sequences of SEQ IDNO: 276, 244, 218, 228, 162, 50, 74, 44, and 116 were subcloned into thepET28b expression vector (Novagen, Madison, Wis.) with N-terminalHis-tags to allow for purification. SEQ ID NO:275 is the sequence of thegene that encodes the aldolase having the amino acid sequence of SEQ IDNO:276. SEQ ID NO:243 is the sequence of the gene that encodes thealdolase having the amino acid sequence of SEQ ID NO:244. SEQ ID NO:217is the sequence of the gene that encodes the aldolase having the aminoacid sequence of SEQ ID NO:218. SEQ ID NO:227 is the sequence of thegene that encodes the aldolase having the amino acid sequence of SEQ IDNO:228. SEQ ID NO:161 is the sequence of the gene that encodes thealdolase having the amino acid sequence of SEQ ID NO:162. SEQ ID NO:49is the sequence of the gene that encodes the aldolase having the aminoacid sequence of SEQ ID NO:50. SEQ ID NO:73 is the sequence of the genethat encodes the aldolase having the amino acid sequence of SEQ IDNO:74. SEQ ID NO:43 is the sequence of the gene that encodes thealdolase having the amino acid sequence of SEQ ID NO:44. SEQ ID NO:115is the sequence of the gene that encodes the aldolase having the aminoacid sequence of SEQ ID NO:116.

The primers used for cloning are shown in Table 24.

TABLE 24 Aldolase DNA SEQ ID NO: 5′ primer 3′ primer 275 5′-5′-ATAAGAGGATCCTTATTCCTCGG ATAAGACATATGCCTATCGTTGTTAC GCAGCCGCTC-3′ (SEQID NO: 340) GAAG-3′ (SEQ ID NO: 339) 243 5′- 5′-ATAAGACATATGAACAGACCTGTGG ATAAGAGGATCCTTACAGGTACT TTGTC-3′ (SEQ ID NO:341) TGAGACCGAG-3′ (SEQ ID NO: 342) 217 5′- 5′-ATAAGACATATGAGCGTGGTCATCC ATAAGAGGATCCTTACTTCGCTTT GAAAC-3′ (SEQ ID NO:343) GTTATAGGC-3′ (SEQ ID NO: 344) 227 5′- 5′- ATAAGACATATGAACAAGCCCGTGGATAAGAGGATCCTTACAAGTACT TTGTG-3′ (SEQ ID NO: 345) TGAGACCGAGG-3′ (SEQ IDNO: 346) 161 5′- 5′- ATAAGACATATGAGCGTGGTCGTCA ATAAGAGGATCCTTAGCCGTTTTTCCGG-3′ (SEQ ID NO: 347) CCCGTCGGTG-3′ (SEQ ID NO: 348) 49 5′- 5′-AGAAGACATATGATGAGCATCGTCG AGAAGAGGATCCTCAGACATATT TCCAGAAC-3′ (SEQ IDNO: 349) TCAGGCCCTTG-3′ (SEQ ID NO: 350) 73 5′- 5′-AGAAGACATATGATGAGCGTGGTCA ACAACAGGATCCCTATTTCTTCTC TCACC-3′ (SEQ ID NO:351) CGGCGTTTC-3′ (SEQ ID NO: 352) 43 5′- 5′-ATAATAGGATCCTTAGACATATTATAATACATATGAGCGTCGTCGTTC TGAGCCCCTTC-3′ (SEQ ID NO: 354) AGAAC-3′ (SEQID NO: 353) 115 5′- 5′- AGAAGACATATGATGTCGGTTGTCGTAGAAGAGGATCCTCAGATATACT TCAGAAC-3′ (SEQ ID NO: 355) TCAGGCCC-3′ (SEQ IDNO: 356)

The genes encoding the above aldolases were amplified by PCR anddigested with appropriate enzymes (Nde I and BamH I) and gel purified(QIAquick® Gel extraction Kit (Qiagen, Valencia, Calif.)). The digestswere individually ligated into pET28 that had been digested with Nde Iand BamH I and gel purified. The ligation was transformed into TOP10cells (Invitrogen, Carlsbad, Calif.). Miniprep DNA from individualcolonies was analyzed for the presence of inserts by size analysis usingagarose gel electrophoresis. Isolates with an insert were submitted forDNA sequence analysis (Agencourt, Beverly, Mass.).

Purification of Aldolases

Confirmed aldolase clones were transformed into either BL21(DE3) orBL21(DE3) pLysS. Induction was carried out overnight in Terrific Brothat 30° C. Overnight cultures grown with the appropriate antibiotic werediluted into fresh medium (typically 1:100) and grown to an OD₆₀₀˜0.6with aeration at 37° C. Cultures were then induced with 1 mM IPTG andshifted to 30° C. (with aeration) and incubation was continuedovernight. Cells were harvested by centrifugation. The cell pellet wastypically subjected to one freeze thaw cycle to assist with cell lysis.The cell pellet was lysed in BugBuster and Benzonase Nuclease (Novagen,Madison, Wis.) (according to the manufacturer's protocol). Cell debriswas removed by centrifugation. The crude protein extract was applied toa 10 mg capacity HIS-Bind column (Novagen, Madison, Wis.) that had beenprepared according to the manufacturer's protocol. The column was washedand the protein was eluted according to the manufacturer's protocol. Thepurified protein was desalted with PD-10 columns (GE Healthcare,Piscataway, N.J.) and eluted in 50 mM potassium phosphate buffer, pH7.5, containing 4 mM MgCl₂, 200 mM NaCl. Purified protein wasconcentrated with Amicon centrifugal concentrators (5000 MW cutoff)(Millipore, Billerica, Mass.). After concentration, it was noted thatthe aldolases of SEQ ID NO: 44, SEQ ID NO: 28, and SEQ ID NO: 276 showedsome level of precipitation, although the activity was still quite high.Proteins were stored at −80° C. until assayed.

Protein assays were done using the Pierce BCA kit (Pierce, Rockford,Ill.) and the microtiter plate protocol with Bovine Serum Albumin(“BSA”) as the protein standard. The Experion Pro260 electrophoresissystem (Bio-Rad, Hercules, Calif.) or SDS-PAGE was used to estimate thepercentage of aldolase in the purified sample, and to evaluateexpression levels in the soluble cell extract and in total protein.

Testing of Purified Aldolases

Purified aldolases were tested for their ability to produce R,R monatinfrom D-tryptophan, and were compared to the aldolases of SEQ ID NO:28and SEQ ID NO:54 prepared in the same manner. Assays were run inmicrocentrifuge tubes (in duplicate) with purified protein, using thesame concentration of enzyme per assay (50 μg/mL) with the exception ofSEQ ID NO:244, for which 25 μg/mL was used. SEQ ID NO:243 did notexpress well and yielded smaller amounts of purified protein. Two mg/mLof BioCatalytics AT-103 (BioCatalytics, Pasadena, Calif.) was used asthe D-aminotransferase. The following were added per 1 mL of reactionmixture: aldolase, 4 mM MgCl₂, 50 mM D-tryptophan, D-aminotransferase,200 mM sodium pyruvate, 100 mM potassium phosphate buffer pH 7.5, and0.05 mM PLP. Samples were incubated at 30° C. Thirty minute, 1 hour, 3hour, and overnight (19 hour) samples were taken. Table 25 shows theaveraged results of total monatin produced at each time point and the %R,R monatin produced, as determined by reversed phase peak areas. Incolumn 3 of Table 25, additional FDAA-derivatization LC/MS/MS analysisas described in Example 1 was done for some of the reactions and thoseresults are shown in the parentheses.

TABLE 25 Total monatin produced from D-tryptophan and % R,R Aldolase(hr) Total monatin (ppm) % R,R monatin SEQ ID NO: 28 (0.5) 16 99.1 SEQID NO: 28 (1) 53.2 99.2 (99.0) SEQ ID NO: 28 (3) 207.8 98.6 (98.1) SEQID NO: 28 (19) 544.9 95.3 (93.2) SEQ ID NO: 44 (0.5) 46.2 88.0 SEQ IDNO: 44 (1) 92.5 86.8 SEQ ID NO: 44 (3) 319.7 76.4 SEQ ID NO: 44 (19)762.9 67.1 SEQ ID NO: 54 (0.5) 35.3 96.2 SEQ ID NO: 54 (1) 82.7 96.1 SEQID NO: 54 (3) 280.1 92.9 SEQ ID NO: 54 (19) 715.3 77.1 SEQ ID NO: 74(0.5) 51.1 92.6 SEQ ID NO: 74 (1) 89.3 94.3 SEQ ID NO: 74 (3) 269.5 89.9SEQ ID NO: 74(19) 701.9 76.2 SEQ ID NO: 50 (0.5) 55.9 96.7 SEQ ID NO: 50(1) 96.5 96.2 SEQ ID NO: 50 (3) 272.2 95.6 SEQ ID NO: 50 (19) 645.8 88.5SEQ ID NO: 162 (0.5) 37.3 95.7 SEQ ID NO: 162 (1) 75.0 97.1 SEQ ID NO:162 (3) 261.0 95.9 SEQ ID NO: 162 (19) 633.1 87.0 SEQ ID NO: 276 (0.5)37.8 98.8 SEQ ID NO: 276 (1) 71.2 99.3 (99.5) SEQ ID NO: 276 (3) 245.299.0 (99.0) SEQ ID NO: 276 (19) 585.4 96.7 (96.1) SEQ ID NO: 244 (0.5)30.2 97.7 SEQ ID NO: 244 (1) 46.4 98.3 (99.2) SEQ ID NO: 244 (3) 16598.4 (98.7) SEQ ID NO: 244 (19) 572.5 95.6 (93.7) SEQ ID NO: 228 (0.5)52 95.0 SEQ ID NO: 228 (1) 81.7 96.5 SEQ ID NO: 228 (3) 251 95.9 SEQ IDNO: 228 (19) 723 87.1 no aldolase (0.5) 0 no aldolase (1) 0 no aldolase(3) 0.6 58.3 no aldolase (19) 6.5 61.5

The SEQ ID NO:276 aldolase maintained a high level of activity, as wellas the highest stereospecificity for production of R,R monatin. Storageof this enzyme in a buffer omitting the sodium chloride appears toreduce the level of precipitation noted earlier. Magnesium concentrationin the storage buffer does not appear to have an impact on the level ofprecipitation.

The aldolases of SEQ ID NO:276, SEQ ID NO: 28, and SEQ ID NO: 244 alldemonstrated high activity and high stereoselectivity for production ofR,R monatin. These three enzymes were prepared as described in Example23 and assayed anaerobically, as described in Example 24, using 10 mLserum vials. Seven mL assays were done using 0.05 mg/mL of each aldolase(purified) and 2 mg/mL of purified B. sphaericus D-aminotransferaseprepared as described in Example 24. The activity of each aldolase inproduction of monatin from D-tryptophan was compared to the S. melilotiHMG aldolase prepared as described in Example 27. Total monatin wasestimated using the LC-OPA method described in Example 1. The percentageof R,R monatin formed was determined using the FDAA derivatizationmethod described in Example 1. The results are shown below in Table 26.

TABLE 26 Aldolase Time (hr) Monatin (g/L) % R,R formed S. meliloti 233.9 82.0 SEQ ID NO: 28 23 4.0 84.6 SEQ ID NO: 276 23 4.0 95.7 SEQ ID NO:244 23 3.7 88.8 S. meliloti 47 4.5 76.2 SEQ ID NO: 28 47 4.3 84.6 SEQ IDNO: 276 47 4.3 93.2 SEQ ID NO: 244 47 4.5 85.2

These results demonstrate that the aldolase of SEQ ID NO: 276 produceshigh levels of R,R monatin in larger volume anaerobic reactions as well.

Example 23 Expression and Purification of the SEQ ID NO:276 Aldolase

The cloning of the E. coli BL21(DE3)pLysS host carrying the aldolasegene listed as SEQ ID NO:275 on the pET28b plasmid is described above inExample 22.

The SEQ ID NO:276 aldolase with an amino-terminal HIS₆-purification tagwas produced using the EMD Biosciences Overnight Express System II(Novagen, Madison, Wis.) (solutions 1-6) containing 50 μg/mL kanamycinin shake flasks. This expression system induces the expression ofIPTG-inducible systems without the need to monitor cell growth. Afterinoculation of 200-mL aliquots of the medium (in 1 L flasks) from eitherliquid cultures or plates of the aldolase construct, the cultures wereincubated at 30° C. overnight with shaking at 225 rpm. When theOD_(600nm) had reached a minimum of 6, the cells were harvested bycentrifugation and washed once with buffer.

To prepare cell free extract containing the aldolase, the cells weresuspended in 3-4 volumes of 100 mM potassium phosphate, pH 7.8 and thendisrupted using a Microfluidics homogenizer (Microfluidics, Newton,Mass.) (3 passes at 18,000 psi), maintaining the temperature of thesuspension at less than 15° C. Alternatively, cell free extract wasprepared using EMD Biosciences BugBuster® (primary amine-free)Extraction Reagent (Novagen, Madison, Wis.) containing 1 μL/mLBenzonase® Nuclease, 5 μL/mL Protease Inhibitor Cocktail Set II, and0.033 μL/mL rLysozyme™ following the manufacturer's protocol. Allsubsequent purification steps were carried out at 4° C. The cellsuspension was centrifuged for 20-30 minutes at 15,000-20,000×g toremove the cell debris. A 20-25 mL aliquot of the cell free extract wasapplied to a 45 mL column of GE Healthcare Chelating Sepharose™ FastFlow resin (nickel (II) form) (GE Healthcare, Piscataway, N.J.) that hadbeen previously equilibrated with 100 mM potassium phosphate containing200 mM sodium chloride. To generate the nickel form of the resin, theresin was washed with 150 mL of 200 mM nickel (II) sulfate hexahydrateand then with 150 mL of distilled water. After loading the sample, thecolumn was washed/eluted with 150 mL of the equilibration buffercontaining 25 mM imidazole, 150 mL of the equilibration buffercontaining 50 mM imidazole and 150 mL of the equilibration buffercontaining 500 mM imidazole. The HIS₆-tagged protein eluted in the lastwash. The 500 mM imidazole wash was concentrated with Millipore/AmiconCentricon Plus-70 centrifugal filter devices (MWCO 10 kDa) (Millipore,Billerica, Mass.) to 15-20 mL according to the manufacturer'sinstructions. The imidazole and sodium chloride were removed by passagethrough disposable GE Healthcare PD10 columns (GE Healthcare,Piscataway, N.J.) (2.5 mL sample per column) previously equilibratedwith 100 mM potassium phosphate, pH 7.8. The purified aldolase waseluted with 3.5 mL per column of the same buffer.

The protein concentration of each fraction was determined using thePierce BCA assay kit (Pierce, Rockford, Ill.) using BSA as the proteinstandard. The purity of each fraction and the level of expression in thecell free extract were determined using a Bio-Rad Experionmicrocapillary chip system (Bio-Rad, Hercules, Calif.) or using Bio-Rad4-15% SDS-polyacrylamide gradient gels run in a Mini PROTEAN° 3 cellapparatus (Bio-Rad, Hercules, Calif.). The protein was visualized in thepolyacrylamide gels using Bio-Rad Bio-Safe G-250 Coomassie stain(Bio-Rad, Hercules, Calif.) and destained with water. Typically thisprocedure produces˜50 mg of enzyme from 400 mL of overnight culture thatis 85-95% pure as judged by the Experion software. Aliquots (1-5 mL) ofthe purified enzyme were stored at −80° C. until use. Preparation of theenzyme in this manner reduced the level of precipitation of the enzymepreviously noted. The presence of magnesium in the storage buffer had noeffect on the level of precipitation.

Example 24 Production of R,R-monatin Using the SEQ ID NO:276 Aldolase:Optimization of Reaction Conditions

The Bacillus sphaericus (ATCC strain 10208) D-alanine aminotransferasecloned in Example 7 was purified as the HIS₆-tagged protein as describedbelow using EMD Biosciences Overnight Express System II (Novagen,Madison, Wis.) for growth and induction. The EMD Biosciences OvernightExpress System II (solutions 1-6) (Novagen, Madison, Wis.) contained 50μg/mL kanamycin in shake flasks. This expression system induces theexpression of IPTG-inducible systems without the need to monitor cellgrowth. After inoculation of 200-mL aliquots of the medium (in 1 Lflasks) from either liquid cultures or plates of the aminotransferaseconstruct, the cultures were incubated at 30° C. overnight with shakingat 225 rpm. When the OD_(600nm) had reached a minimum of 6, the cellswere harvested by centrifugation and washed once with buffer.

To prepare cell free extract containing the D-alanine aminotransferase,the cells were suspended in 3-4 volumes of 100 mM potassium phosphate,pH 7.8, containing 50 μM pyridoxal phosphate (PLP) and then disruptedusing a Microfluidics homogenizer (Microfluidics, Newton, Mass.) (3passes at 18,000 psi), maintaining the temperature of the suspension atless than 15° C. Alternatively, cell free extract was prepared using EMDBiosciences BugBuster® (primary amine-free) Extraction Reagent (Novagen,Madison, Wis.) containing 1 μL/mL Benzonase® Nuclease, 5 μL/mL ProteaseInhibitor Cocktail Set II, and 0.033 μL/mL rLysozyme™ following themanufacturer's protocol. All subsequent purification steps were carriedout at 4° C. The cell extract was centrifuged for 20-30 minutes at15,000×g to remove the cell debris. A 20-25 mL aliquot of the cell freeextract was applied to a 45 mL column of GE Healthcare ChelatingSepharose™ Fast Flow resin (nickel (II) form) (GE Healthcare,Piscataway, N.J.) that had been previously equilibrated with 100 mMpotassium phosphate containing 200 mM sodium chloride and 50 μM PLP. Togenerate the nickel form of the resin, the resin was washed with 150 mLof 200 mM nickel (II) sulfate hexahydrate and then with 150 mL ofdistilled water. After loading the sample, the column was washed/elutedwith 150 mL of the equilibration buffer containing 25 mM imidazole, 150mL of the equilibration buffer containing 50 mM imidazole and 150 mL ofthe equilibration buffer containing 500 mM imidazole. The HIS₆-taggedprotein eluted in the last wash. The 500 mM imidazole wash wasconcentrated with Millipore/Amicon Centricon Plus-70 centrifugal filterdevices (MWCO 10 kDa) (Millipore, Billerica, Mass.) to 15-20 mLaccording to the manufacturer's instructions. The imidazole and sodiumchloride were removed by passage through disposable GE Healthcare PD10columns (GE Healthcare, Piscataway, N.J.) (2.5 mL sample per column)previously equilibrated with 100 mM potassium phosphate, pH 7.8containing 0.5 μM PLP. The purified aminotransferase was eluted with 3.5mL per column of the same buffer.

The protein concentration of each fraction was determined using thePierce BCA assay kit (Pierce, Rockford, Ill.) with BSA as the proteinstandard. The purity of each fraction and the level of expression in thecell free extract fraction were determined using a Bio-Rad Experionmicrocapillary chip system (Bio-Rad, Hercules, Calif.) or using Bio-Rad4-15% SDS-polyacrylamide gradient gels (Bio-Rad, Hercules, Calif.) runin a Mini PROTEAN® 3 cell apparatus. The protein was visualized in thepolyacrylamide gels using Bio-Rad Bio-Safe G-250 Coomassie stain(Bio-Rad, Hercules, Calif.) and destained with water. Typically thisprocedure produces˜50 mg of enzyme from 200 mL of overnight culture thatis 85-90% pure as judged by the Experion software or from analysis ofthe SDS-PAGE gels. Aliquots (1-5 mL) of the purified enzyme were storedat −80° C. until use.

The SEQ ID NO:276 aldolase (cloned in Example 22) was purified as theHIS₆-tagged protein as described in Example 23.

The preferred metal cofactor for the SEQ ID NO:276 aldolase wasdetermined by screening a variety of divalent metals. The reactions wereset up anaerobically in 10 mL serum bottles with 7 mL final volumes. Abulk solution consisting of 100 mM potassium phosphate (pH 7.8), 200 mMsodium pyruvate, 0.05 mM PLP and 0.01% (v/v) Tween 80 was prepared to afinal volume of 48.8 mL and sparged with nitrogen for 30 minutes.D-Tryptophan (143 mg; final concentration of 100 mM) was dispensed intoseven 10 mL serum vials. To each of the vials was added 0.014 mL of a 2M stock solution of a divalent metal cation, prepared from the chloridesalt (final concentration of 4 mM). For the negative control, 0.014 mLof dH₂O was added. The serum vials were capped with rubber septa andsparged with nitrogen via 16-18 gauge needles. Under anaerobicconditions, 5.625 mL of the anaerobic bulk solution was added to eachanaerobic serum bottle. Subsequently, the B. sphaericus D-alanineaminotransferase and the SEQ ID NO:276 aldolase were added anaerobicallyto each serum bottle to a final concentration of 2 mg/mL and 0.05 mg/mL,respectively. The solutions were incubated at room temperature withgentle mixing for 18 hours. Final monatin was analyzed according to themethods described in Example 1 using the Liquid Chromatography-PostColumn Fluorescence Detection of Amino Acids method. (Table 27).

TABLE 27 Metal Cofactor Final Monatin (mM) at 18 h None (negativecontrol) 1.7 Magnesium 10.6 Manganese 10.0 Cobalt 6.7 Zinc 4.9 Nickel1.5 Calcium 0.7

The reaction conditions for the SEQ ID NO:276 aldolase were furtherinvestigated with a two-level fractional factorial experiment designedusing the statistical software Design Expert 7.0.0 (Stat-Ease, Inc.;Minneapolis, Minn.). The screening design consisted of a single block offive factors at two levels with four centerpoints (20 runs total). Thefive factors to be optimized were the metal cofactor concentration,reaction pH, Tween® 80 concentration, pyruvate to tryptophan ratio, andthe aldolase concentration (Table 28).

Conical polypropylene tubes (14 mL) containing 143 mg of D-tryptophanwere de-oxygenated in an anaerobic glove box (Coy Laboratory Products,Inc; Grass Lake, Mich.) overnight. Stock solutions of 2 M MgCl₂; 1Mpotassium phosphate at pH 7.0, 7.75, and 8.5; 10% (v/v) Tween® 80; 2 Msodium pyruvate, and 10 mM PLP (pyridoxal 5′-phosphate) were prepared indegassed water and equilibrated in the anaerobic glove box overnight.Stock solutions of purified B. sphaericus D-alanine aminotransferase andthe SEQ ID NO:276 aldolase were thawed on ice and used in the anaerobicglove box immediately. Stock solutions were added to the 14 mL conicaltubes containing the D-tryptophan to obtain the concentrationsdetermined by the statistical design (Table 28). Degassed water wasadded to each tube to bring the final volume, along with the enzymeadditions, to 7.0 mL. The tubes were incubated at room temperature inthe anaerobic glove box with gentle mixing for up to 24 hours. Monatinconcentration and isomeric purity were analyzed according to the methodsdescribed in Example 1 using the Liquid Chromatography-Post ColumnFluorescence Detection of Amino Acids method and the LC/MS/MS MultipleReaction Monitoring for the Determination of the StereoisomerDistribution of Monatin in in vitro and in vivo Reactions method (FDAAderivatization method).

TABLE 28 Aldolase of SEQ ID Mg Tween ® NO: 276 Run # std # Block (mM) pH(%) Pyr:Trp (mg/mL) 20 1 Block 1 5.00 7.75 0.01 2.00 0.05 8 2 Block 19.00 8.50 0.02 1.00 0.01 3 3 Block 1 1.00 8.50 0.00 1.00 0.01 16 4 Block1 9.00 8.50 0.02 3.00 0.09 7 5 Block 1 1.00 8.50 0.02 1.00 0.09 12 6Block 1 9.00 8.50 0.00 3.00 0.01 6 7 Block 1 9.00 7.00 0.02 1.00 0.09 28 Block 1 9.00 7.00 0.00 1.00 0.01 15 9 Block 1 1.00 8.50 0.02 3.00 0.014 10 Block 1 9.00 8.50 0.00 1.00 0.09 5 11 Block 1 1.00 7.00 0.02 1.000.01 1 12 Block 1 1.00 7.00 0.00 1.00 0.09 13 13 Block 1 1.00 7.00 0.023.00 0.09 14 14 Block 1 9.00 7.00 0.02 3.00 0.01 17 15 Block 1 5.00 7.750.01 2.00 0.05 11 16 Block 1 1.00 8.50 0.00 3.00 0.09 18 17 Block 1 5.007.75 0.01 2.00 0.05 9 18 Block 1 1.00 7.00 0.00 3.00 0.01 19 19 Block 15.00 7.75 0.01 2.00 0.05 10 20 Block 1 9.00 7.00 0.00 3.00 0.09

Statistical analysis of the data indicated that reaction pH,pyruvate:tryptophan ratio and aldolase concentration were thesignificant factors affecting monatin titer, isomeric purity and carbonyield. A desirability graph was generated using the Design Expertsoftware in which the factors were varied in order to maximize the goalsof highest monatin titer and highest isomeric purity under conditions ofexcess pyruvate. The reactions conditions indicated as optimum were 1 mMMgCl₂, pH>8.0, 0.01% (v/v) Tween® 80, and 0.01 mg/mL SEQ ID NO:276aldolase. This is a 5 fold reduction in the typical amount of aldolaseutilized, as well as a 4-fold reduction in the amount of divalent metaltypically used.

Additional experiments were performed to determine the optimum pH rangefor the reaction process. Stock solutions of 1M EPPS buffer wereprepared at increments of 0.2 pH units between pH 7.0 and 9.0. Thesesolutions were degassed and equilibrated in the anaerobic glove boxovernight. Polypropylene tubes (14 mL) containing 143 mg of D-tryptophanwere de-oxygenated in an anaerobic glove box overnight. Stock solutionsof 2M MgCl₂, 10% (v/v) Tween® 80, 2M sodium pyruvate and 10 mM PLP wereprepared in degassed water and equilibrated in the anaerobic glove box.Preparations of purified B. sphaericus D-alanine aminotransferase andSEQ ID NO:276 aldolase were thawed on ice and used immediately in theanaerobic glove box. The stock solutions were added to the 14 mL conicaltubes to give a final concentration of 100 mM EPPS, 200 mM pyruvate, 100mM tryptophan, 1 mM MgCl₂, 0.01% (v/v) Tween® 80, 0.05 mM PLP, 2 mg/mLB. sphaericus D-alanine aminotransferase, and 0.01 mg/mL SEQ ID NO:276aldolase in a total volume of 7 mL per tube. The reactions wereincubated at room temperature in the anaerobic glove box with gentleagitation for 22 hours. Samples were removed and analyzed for monatin asdescribed in Example 1 using the LC/MS/MS multiple reaction monitoringmethod (Table 29).

TABLE 29 Reaction pH Monatin (mM) at 22 h 7.0 5.8 7.2 9.9 7.4 7.8 7.610.6 7.8 14.0 8.0 14.2 8.2 14.3 8.4 12.6 8.6 12.3 8.8 10.8 9.0 11.1

The results indicated that monatin formation increased with increasingpH between 7.0-8.0. Monatin formation reached a maximum in the range ofpH 8.0-8.2, and decreased above pH 8.4. Additionally, the isomericpurity of monatin decreased above pH 8.4.

Further reaction optimization was done using the aldolase of SEQ ID NO:276 (0.01 mg/mL), 2 mg/mL of the T243N 4978 DAT (untagged, from Example26), 1 mM MgCl₂, 200 mM sodium pyruvate, 0.05 mM PLP, 0.01% Tween-80,and 100 mM D-tryptophan at pH 8.5. The cells used to produce thealdolase and the D-aminotransferase were broken open in 50 mM EPPS, pH8.4 using a Microfluidics homogenizer (Microfluidics, Newton, Mass.) (3passes at 20,000 psi). The cell debris was removed by centrifugation(20,000×g for 30 minutes) and the clarified cell extracts were used inthe enzymatic reactions. No additional buffer was utilized, but thereaction mixtures were adjusted to pH 8.5 using sodium hydroxide andflushed with nitrogen prior to addition of enzyme. Two-hundred fifty mLreactions were carried out in 0.7 L Sixfor agitated fermenters (InforsAG, Bottmingen, Switzerland) at 30° C. using nitrogen in the headspace.Potassium phosphate was added to a final concentration of 0, 5, 10, 20,and 50 mM. The addition of 5-10 mM phosphate was found to be optimal,producing 3.5 g/L monatin (quantitated by LC/MS/MS as described inExample 1).

Example 25 Cloning of A Novel Bacillus D-Amino Acid Aminotransferase

A Bacillus D-amino acid aminotransferase (“DAAT” or “DAT”) (EC 2.6.1.21,also known as D-alanine aminotransferase or D-aspartateaminotransferase) was produced recombinantly. This aminotransferase wasused in coupled assays with aldolases for production of R,R monatin.This aminotransferase enzyme is homologous to D-aminotransferasesdescribed previously for production of monatin (U.S. PublishedApplication No. 20040063175 and U.S. Published Application No.20050282260). The organism ATCC 4978—Bacillus sphaericus originallydeposited as Bacillus rotans—was ordered from the ATCC and used toprepare genomic DNA. Degenerate primers were designed in the regions ofprotein sequence conservation of known Bacillus D-aminotransferases andused for polymerase chain reaction (“PCR”) amplification of internalDAAT gene sequence from the ATCC strain mentioned above. Genome walkingwas performed using the BD GenomeWalker™ Universal Kit (Clontech,Mountain View, Calif.). Sequence analyses (Agencourt BioScienceCorporation, Beverly, Mass.) verified a full-length coding sequence forthe DAAT gene from ATCC 4978. The DNA sequence of the DAAT gene fromATCC 4978 is SEQ ID NO:357 and is shown below. The gene of SEQ ID NO:357may be amplified by standard PCR protocols and cloned using standardrecombinant DNA techniques. The gene of SEQ ID NO:357 may also bereconstructed by any method known to a person of ordinary skill in theart, such as assembly PCR methods known to one skilled in the art.

The ATCC 4978 DAAT DNA sequence is:

(SEQ ID NO: 357) atgagttata gcttatggaa tgaccaaatt gtgaatgatg aagaagtagtagttgataag gaggaccgtg gctatcaatt tggcgatggt gtttatgaag ttgtaaaagtatataacggt gaattattta cagcggagga gcatgtcgat cgtttttacg cgagtgctgaaaaaattcgc gttacgatcc cttatacaaa agacaaattg catcaattat tgcatcagttagttgaaatg aataaagttc aaacaggaca tatttatttc caaattacgc gtggtgcaggccctcgtaat catattttcc ctggtgatga agtgaagcca gtattaacag gtaataccaaggaaaatcca cgtcccgtag caaactttga aaaaggtgtg aaagcaacat ttgtagaagacattcgttgg ttacgctgtg acattaaatc attaaattta cttggtgcgg tacttgctaaacaagaagca catgaaaaag gatgctatga agcggtttta catcgtgatg aaatcgtaacagaaggctct tcttcaaata tttatggaat taaagatggc gtattataca cacatccagcgaataacttc atcttaaatg gtattacacg tcaagtaatc attaaatgtg ctgctgaaattggcttacca gtgaaggaag aagcaatgac aaaaactcag cttcttgcaa tggatgaagtgattgtttca tcaacgactt cagaagtaac gccaattatc gacatagatg gaacagtaattggtgcgggt aaaccgggtg actggacacg taaattacaa gcacaatttg atacgaaaatcccaaaaggt attcgcgcat aa

The amino acid sequence of the DAAT gene from ATCC 4978 as encoded bythe above DNA sequence is SEQ ID NO:358 and is shown below:

(SEQ ID NO: 358) Met Ser Tyr Ser Leu Trp Asn Asp Gln Ile Val Asn Asp GluGlu Val Val Val Asp Lys Glu Asp Arg Gly Tyr Gln Phe Gly Asp Gly Val TyrGlu Val Val Lys Val Tyr Asn Gly Glu Leu Phe Thr Ala Glu Glu His Val AspArg Phe Tyr Ala Ser Ala Glu Lys Ile Arg Val Thr Ile Pro Tyr Thr Lys AspLys Leu His Gln Leu Leu His Gln Leu Val Glu Met Asn Lys Val Gln Thr GlyHis Ile Tyr Phe Gln Ile Thr Arg Gly Ala Gly Pro Arg Asn His Ile Phe ProGly Asp Glu Val Lys Pro Val Leu Thr Gly Asn Thr Lys Glu Asn Pro Arg ProVal Ala Asn Phe Glu Lys Gly Val Lys Ala Thr Phe Val Glu Asp Ile Arg TrpLeu Arg Cys Asp Ile Lys Ser Leu Asn Leu Leu Gly Ala Val Leu Ala Lys GlnGlu Ala His Glu Lys Gly Cys Tyr Glu Ala Val Leu His Arg Asp Glu Ile ValThr Glu Gly Ser Ser Ser Asn Ile Tyr Gly Ile Lys Asp Gly Val Leu Tyr ThrHis Pro Ala Asn Asn Phe Ile Leu Asn Gly Ile Thr Arg Gln Val Ile Ile LysCys Ala Ala Glu Ile Gly Leu Pro Val Lys Glu Glu Ala Met Thr Lys Thr GlnLeu Leu Ala Met Asp Glu Val Ile Val Ser Ser Thr Thr Ser Glu Val Thr ProIle Ile Asp Ile Asp Gly Thr Val Ile Gly Ala Gly Lys Pro Gly Asp Trp ThrArg Lys Leu Gln Ala Gln Phe Asp Thr Lys Ile Pro Lys Gly Ile Arg Ala

The novel D-aminotransferase obtained from strain ATCC 4978 has aprotein sequence that has distinct amino acid residue changes whencompared to the B. sphaericus ATCC 10208 published D-aminotransferasesequence. The DAAT from ATCC 4978 has only 72% identity with the DAATfrom B. sphaericus (ATCC 10208). While this strain is currently listedas B. sphaericus in the ATCC, it was deposited as B. rotans. Based onthe sequence alignments and the highlighted differences between thisnovel DAAT and the DAAT from B. sphaericus, a number of candidateresidues are identified that can be evaluated for their role(individually or in combination) in increasing DAAT activity for R,Rmonatin biosynthesis, in these, as well as other DAAT sequences.

Example 26 Characterization of Mutants of D-Aminotransferase from ATCC4978 Experimental Overview

The novel D-aminotransferase gene (described in Example 25) fromBacillus strain ATCC 4978 was mutagenized using site-directed methods.The mutant genes were expressed and assayed for activities of interestfor monatin production pathways, especially when coupled with one ormore aldolases.

In addition to the ideas listed in Example 16 for site directedmutagenesis targets, other ideas were developed by the actual docking ofR-MP into the active site of the YM-1 crystal structure primary aminoacid sequence alignments were used to determine if the 4978aminotransferase protein was likely to have similar structuralcharacteristics in that region. It was expected that the followingadditional mutations would be beneficial (using the 4978aminotransferase's amino acid numbering). It was thought thatmutagenesis of alanine 153 to arginine would stabilize the secondcarboxyl group of the substrate (R-MP). This change is likely toincrease steric hindrance, so to compensate, the serine residues atpositions 181 and 182 were changed to alanine or glycine. It was alsohypothesized that one could introduce an arginine at position 180, 181,or 182 and convert one or more of the other serine residues to alanineor glycine to make room for the bulkier side chain of arginine. Thephenylalanine at amino acid 200 is spatially close to where R-MP ispredicted to dock into the active site and there is a large amount ofvariability in this residue amongst the D-aminotransferases thatcatalyze monatin transamination fairly well. It was thought that aminoacid modifications at this position could be useful. Mutation of leucine151 to phenylalanine was predicted to potentially improve interactionswith the indole ring of the substrate.

Based upon literature, it was hypothesized that mutation of threonine243 to asparagine may improve R-MP selectivity for transaminationreactions. Likewise, it was thought that mutagenesis of asparagine 100to alanine may improve the specific activity of the enzyme for monatintransamination reactions (Ro, et al., FEBS Lett 398:141-145, (1996);Sugio, S, et al., Biochemistry 34:9661-9669, (1995); EP1580268).

Lee et al. characterized mutants of the 141-144 region (loop) and foundthat D-aminotransferases with the EYcY rather than the LRcD (which isnative to our protein) tend to have a lower Km for dicarboxylic acidsubstrates. (Lee S G, Hong S P, Song J J, Kim S J, Kwak M S, Sung M H.Functional and structural characterization of thermostable D-amino acidaminotransferases from Geobacillus spp. Appl Environ Microbiol. 2006February; 72(2):1588-94). Because MP is a dicarboxylic acid substrate,similar to alpha-keto glutarate, and the concentrations of MP are fairlylow in a typical monatin production reaction mixture, a decreased K_(m)could potentially help the activity of a mutant DAT for monatinproduction.

Below, methods are described for creating the 4978 D-aminotransferasemutants, as well as assay results using these mutants.

Mutagenesis

The primers for mutagenesis were designed following the suggestionslisted in the Stratagene Multi-Change kit (Stratagene, La Jolla,Calif.). The primers were 5′-phosphorylated. Mutagenesis was done usingthe Stratagene Multi-Change kit (Stratagene, La Jolla, Calif.) followingthe manufacturer's instructions. The templates used for mutagenesis wereeither the pET30 (untagged) or pET28 (tagged) 4978 DAT constructsdescribed in Example 25. The primers are listed below in Table 30:

TABLE 30 Amino Mutant acid Name change Primer 4978-22 T243NGTGATTGTTTCATCAACGAATTCAGAAGTAACG CC (SEQ ID NO: 359) 10 T243RGTGATTGTTTCATCAACGCGTTCAGAAGTAACG CC (SEQ ID NO: 360)  7 T243SGTGATTGTTTCATCAACGAGTTCAGAAGTAACG CC (SEQ ID NO: 361) 19 T243AGTGATTGTTTCATCAACGGCTTCAGAAGTAACG CC (SEQ ID NO: 362) 15 N100AGTGCAGGCCCTCGTGCTCATATTTTCCCTGG (SEQ ID NO: 363) B T243QGAAGTGATTGTTTCATCAACGCAGTCAGAAGT AACGCCAATTATC (SEQ ID NO: 364)  2T243N/ above primers used together N100

E. coli XL10-Gold cells (Stratagene, La Jolla, Calif.) were transformed,and resultant purified plasmid preparations were sequenced to verifythat the correct mutations were incorporated.

Expression and Assay

Plasmid DNA preparations containing the correct mutants or the wildtype4978 DAT were transformed into the E. coli expression host BL21(DE3)(Novagen, Madison, Wis.). The cultures were grown using the protocolsdescribed above, the plasmids were isolated using Qiagen miniprep kit(Qiagen, Valencia, Calif.) and analyzed by restriction digestion, asdescribed above, to confirm the presence of an insert.

Induction of the DAAT gene was typically performed in LB mediumcontaining kanamycin (50 μg/mL). The cells were grown to an OD_(600m) of0.4-0.8 at 37° C., induced with 0.1 mM IPTG (isopropyl thiogalacatoside)and sampled at 3-4 hours post induction.

Cellular extracts were prepared with BugBuster Reagent and BenzonaseNuclease (Novagen, Madison, Wis.). One ml assays were performed at 30°C. with gentle shaking and contained 10.2 mg D-tryptophan, 0.05 mM PLP,4 mM MgCl₂, 100 mM potassium phosphate buffer pH 7.5, approximately 50μg of aldolase, 200 mM pyruvate, and 0.150-0.5 mg/mL D-aminotransferasesupplied as cellular extracts. Total protein assays were done using theBio-Rad total protein kit (Coomassie) (Bio-Rad, Hercules, Calif.) or thePierce BCA kit (Pierce, Rockford, Ill.), and percent expression of theD-aminotransferase was estimated by SDS-PAGE or the Bio-Rad ExperionAutomated Electrophoresis System (Bio-Rad, Hercules, Calif.). Sampleswere taken at 3 hours and overnight.

The R-specific aldolase of SEQ ID NO: 28 was used in assays withapproximately 0.150 mg/mL D-aminotransferase.

The first assays showed that the following mutants (untagged) hadtransamination activity (in order of highest to lowest): T243N, T243S,T243N/N100A, N100A. It was also noted that the T243N appeared to raisethe stereo-purity of the R,R monatin produced. Assays were repeatedusing purified Comamonas testosteroni ProA aldolase (100 μg/ml) and 0.50mg/ml of D-aminotransferase mutants (untagged, supplied as cellularextract). Samples were taken at 2 hours and overnight. The results forthe active proteins are shown below, duplicate results were averaged.The % R,R monatin was determined by peak area on reversed phase HPLC,and then measured using the FDAA derivatization method described inExample 1. In column 3 of Table 31, additional FDAA-derivatizationLC/MS/MS analysis as described in Example 1 was done for some of thereactions and those results are shown in the parentheses. Only R,R andS,R monatin are produced from D-tryptophan. The T243R mutant did notappear to produce monatin under the conditions tested, and the T243Amutant produced very low levels of monatin.

TABLE 31 Enzyme untagged Total monatin (time - hr or overnight) (ppm) %R,R 4978 wildtype (2 hours) 4.7 41.6 4978 wildtype (overnight) 43.2 35.1(30.9) T243S (2 hours) 55.0 37.4 (21.7) T243S (overnight) 97.7 35.5(29.8) T243N (2 hours) 73.2 86.7 (88.3) T243N overnight 120.9 86.3(86.1) N100A (2 hours) 12.0 40.8 N100A (overnight) 22.3 41 T243A (2hours) 0.8 ~100 T243A (overnight) 1.3 ~100

Although the assays were performed estimating percent D-aminotransferaseusing Bio-Rad Experion software, it is clear that the T243S and T243Nmutants had increased activity compared to the wildtype enzyme. TheT243N mutant also provided an additional benefit of increasingdramatically the % R,R monatin formed. This enzyme has an increasedpreference for R-MP as compared to S-MP in transamination reactions. TheN100A mutant did not increase activity alone or in combination withT243N contrary to what was suggested in the literature. A V34A sitedirected mutant of the untagged 4978 DAT was also created using similarmethods, as described above. The V34A sited directed mutant was found tohave significantly less activity than the wild-type enzyme under theconditions tested.

Another point of interest in the initial assays was that the wildtypeenzyme appeared to have more activity when it was produced with anN-terminal His-tag. Subsequent mutagenesis was done on the taggedversion of the gene. Additionally, the most promising mutants above weresubcloned into pET28b that has an N-terminal His-tag. These werepurified using Novagen HIS-bind columns and the manufacturer's protocolwith the recommended buffers (Novagen, Madison, Wis.). The buffer of theeluent fractions was exchanged, using GE Healthcare PD10 columns (GEHealthcare, Piscataway, N.J.), to the buffer used in the assays

One ml assays with purified D-aminotransferase (0.5 mg/ml) and purifiedR-specific aldolase SEQ ID NO:276 (50 μg/ml) were conducted at 30° C.with gentle shaking and contained 10.2 mg D-tryptophan, 0.05 mM PLP, 200mM pyruvate, 4 mM MgCl₂, and 100 mM potassium phosphate buffer pH 7.5.Duplicate samples were incubated for 2 hours and overnight. As apositive control, the Bacillus sphaericus DAT (cloned in Example 7) wasused in the same assays. The results are presented in Table 32:

TABLE 32 Total Enzyme - tagged monatin (time: hours or overnight) (ppm)% R,R 4978 wildtype (2 hours) 43 98.4 4978 wildtype (overnight) 96.798.3 (95.9) T243N (2 hours) 197.5 100 T243N overnight 301.2 99.9 (99.6)B. sphaericus DAT (2 hours) 58.2 99.7 B. sphaericus DAT (overnight)221.7 98.7 (96.6) T243Q (2 hours) 7.1 100 T243Q (overnight) 12.4 98.8

The data above show that the T243N mutant clearly produces the highestamount of monatin at 2 hours. As time increases, the ratio of T243Nmutant to B. sphaericus DAT positive control is reduced. This resultsuggests that the T243N mutant is not as stable during the monatinreaction as the B. sphaericus DAT. When assayed under similarconditions, the T243S (purified tagged) enzyme had similar levels ofactivity to the T243N mutant; however, the percent R,R monatin producedwas lower (97.2% at both 2 h and overnight). The T243N/N100A mutant hadless activity than the T243N mutant. However, both T243S and T243N/N100Ahad higher activity than the wildtype 4978 DAT.

Transamination assays were performed to determine which reaction rateswere improved when using the T243N mutant in place of the B. sphaericusDAT. One-half mL assays were performed at 30° C. taking time points at 1hour, 2 hours, and 5 hours. The assays contained 25 mM monatin orD-tryptophan, 25 mM pyruvate, 100 mM potassium phosphate pH 7.5, 50 μMPLP, and 0.1 mg D-aminotransferase (tagged, purified). In the case whereless than 100 μg DAT was used, the amount of alanine was normalized to100 μg of D-aminotransferase. Samples were treated with formic acid andanalyzed by LC-OPA for the presence of the coproduct, alanine. Theresults are shown in Tables 33 and 34.

TABLE 33 Transamination activity with R,R monatin as substrate EnzymeD-alanine (mM) wildtype 4978 DAT (2 hr) 0.54 wildtype 4978 DAT (5 hr)1.11 T243N/N100A (2 hr) 1.32 T243N/N100A (5 hr) 2.78 T243S (2 hr) 1.5T243S (5 hr) 2.61 T243N (2 hr) 1.26 T243N (5 hr) 2.65 B. sphaericus DAT(2 hr) 0.97 B. sphaericus DAT (5 hr) 2.2

TABLE 34 Transamination activity with D-tryptophan as substrate EnzymeD-alanine (mM) wildtype 4978 DAT (1 hr) 4.55 wildtype 4978 DAT (2 hr)8.47 T243N/N100A (1 hr) 8.52 T243N/N100A (2 hr) 12.67 T243S (1 hr) 4.89T243S (2 hr) 8.1 T243N (1 hr) 7.19 T243N (2 hr) 10.83 B. sphaericus DAT(1 hr) 8.7 B. sphaericus DAT (2 hr) 12.54

For the D-tryptophan reactions, the results show that some of theenzymes had reached equilibrium at 2 hours. The R,R monatin reactionsare clearly rate-limiting and improvements to this activity have more ofan impact on monatin production rates from D-tryptophan.

Further assays were done to examine the stability of the T243N 4978 DATmutant. The wildtype enzyme also loses activity over time. Example 27describes methods to improve the stability of the T243ND-aminotransferase mutant. When freshly prepared untagged and taggedversions of the T243N mutant are prepared and compared for activity, itwas found that the untagged version had a better temporal stability,making it overall a better version of the enzyme to use in monatinproduction reactions.

Additional mutants of 4978 DAT were made by methods commonly known tothose skilled in the art. However, these mutations all resulted inprotein that was insoluble under the conditions that they were prepared,and thus could not be assayed for activity. The mutations that resultedin insoluble protein were:

S180A/S181A/S182R;

L151F;

V34G

S181R

A153R/S181A/S182A;

A153R/S182A;

A153R/S182G;

S180R/S181A/S182G;

S180R/S181A/S182A;

S180R/S181G/S182G;

S180G/S181R/S182G; and

S180A/S181R/S182A.

Additional Mutagenesis

To create the F200M 4978 DAT mutant, the wildtype 4978 DAT open readingframe from Example 25 (tagged) was amplified with primers 73 and 80(below) and PfuTurbo DNA Polymerase (Stratagene, La Jolla, Calif.) andcloned into pCRII-Blunt (Invitrogen, Carlsbad, Calif.). Its sequence wasverified (Agencourt, Beverly, Mass.). The 5′ and 3′ regions wereamplified using primers 80 and 96 and 99 and 103, respectively. Theamplified DNA was then gel purified using Qiagen QIAquick Gel ExtractionKit (Qiagen, Valencia, Calif.). They amplified DNA was subjected againto PCR using primers 80 and 99. The amplified DNA was gel purified asdescribed above and cloned into pCRII Blunt and its sequence verified.The DAT open reading frame was subcloned as an NdeI/XhoI restrictiondigest fragment into pET28b.

TABLE 35 Primer Number Sequence 73CATATGAGTTATAGCTTATGGAATGACCAAATTGTGAATG (SEQ ID NO: 365) 80CTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTC (SEQ ID NO: 366) 96AATATTTATGGAATTAAAGATGGCGTATTATACACACATCCAGCGAATAACATGATCTTAAATGGTATTACACGTCAAGTAATCATTA AATGTGC (SEQ ID NO: 367) 99GGCCAGTGAATTGTAATACGACTCACTATAGGGC (SEQ ID NO: 368) 103CGCCATCTTTAATTCCATAAATATTTGAAGAAGAGCCTTCTG (SEQ ID NO: 369)

The following primers were designed for additional site-directedmutagenesis using the QuikChange® Multi Site-Directed Mutagenesis Kit(Stratagene, La Jolla, Calif.). Mutagenesis was done using theStratagene Multi-Change kit (Stratagene, La Jolla, Calif.) following themanufacturer's instructions. The template used for mutagenesis was thepET28 (tagged) 4978 DAT construct described in Example 25. A doublemutant was also created using the F200Y mutant as template and doing anadditional round of mutagenesis with the T243N (listed above) primer.

TABLE 36 Mutant Oligo 141-LRcD-144 -> GCAACATTTGTAGAAGACATTCGTTGGGAATACEYcY TGTTACATTAAATCATTAAATTTACTTGGTGCG (SEQ ID NO: 370) F200YGTATTATACACACATCCAGCGAATAACTACATC TTAAATGGTATTACACGTCAAG (SEQ ID NO:371) S244K GCAATGGATGAAGTGATTGTTTCATCAACGACTAAAGAAGTAACGCCAATTATCGACATAGATG (SEQ ID NO: 372) 243-TS-244 -> NKGCAATGGATGAAGTGATTGTTTCATCAACGAAT AAAGAAGTAACGCCAATTATCGACATAGATG (SEQID NO: 373) 243-TS-244 -> NR GCAATGGATGAAGTGATTGTTTCATCAACGAATCGTGAAGTAACGCCAATTATCGACATAGATG (SEQ ID NO: 374)

The mutant coding regions were verified by DNA sequencing (Agencourt).The sequence-verified plasmids were transformed into BL21(DE3) cells(Novagen, Madison, Wis.).

Expression and Assay

Cultures containing 100 ml LB with 50 μg/ml kanamycin in a 500 mlbaffled flask were inoculated with one ml of an overnight culture andgrown at 37° C. to an optical density (at 600 nm) of approximately 0.6.Production of the protein was induced by IPTG at a final concentrationof 1 mM. Cells were incubated at 30° C. for 4.5 hours after the additionof the IPTG. Cells were centrifuged and frozen at −80° C. Cells weredisrupted (prepared using Novagen BugBuster reagent (Novagen, Madison,Wis.) containing 1 μL/mL benzonase nuclease, 5 μL/mL protease inhibitorcocktail II, and 0.033 μL/mL rLysozyme following Novagen's recommendedprotocol) and analyzed by SDS-PAGE. Mutants (141-LRcD-144->EYcY) and(243-TS-244->NR) resulted in insoluble proteins under the conditions inwhich they were prepared. Mutant 243-TS-244->NK did not havequantifiable activity under the conditions tested, and is probably aweak activity enzyme in comparison to wildtype as is the S244K mutant.

His-tagged proteins were purified as follows. HIS-bind columns (Novagen,Madison, Wis.) were equilibrated with 10 mL of 100 mM potassiumphosphate, pH 7.8, containing 200 mM NaCl and 50 μM PLP. Cell-freeextracts were loaded on the column. The columns were washed with 10 mLof equilibration buffer, 10 mL equilibration buffer containing 25 mMimidazole, and 10 mL equilibration buffer containing 50 mM imidazole.Proteins were eluted with 5 ml equilibration buffer containing 500 mMimidazole. Proteins were desalted using PD10 columns which wereequilibrated in 100 mM potassium phosphate, pH 7.8 containing 50 μM PLP.The purified proteins were concentrated and quantified using theBradford Assay (Bio-Rad, Hercules, Calif.).

The D-aminotransferase mutants were assayed using 500 μg/ml of theD-aminotransferase, 50 μg/ml of the aldolase of SEQ ID NO:276, 4 mMMgCl₂, 50 mM potassium phosphate pH 8, 200 mM sodium pyruvate, 0.05 mMPLP and 20.4 mg/ml D-tryptophan for assay conditions. The final volumewas 1.25 ml. Samples (200 μl) were taken after 0.5, 1, 2 and 14 hoursand frozen until the experiment was complete. Samples were filtered,diluted 1 to 10, and analyzed by LC/MS/MS as described in Example 1.

The wildtype 4978 D-aminotransferase from Example 25 was used as areference for percent relative activity. Table 37 shows relativeactivity of each mutant at each time point.

TABLE 37 D-aminotransferase time (hr) % activity 4978 wildtype 0.5 100T243N 0.5 270 F200M 0.5 50 F200Y 0.5 70 F200M/T243N 0.5 183 S244K 0.5 44978 wildtype 1 100 T243N 1 289 F200M 1 55 F200Y 1 81 F200M/T243N 1 203S244K 1 6 4978 wildtype 2 100 T243N 2 266 F200M 2 51 F200Y 2 79F200M/T243N 2 185 S244K 2 6 4978 wildtype 14 100 T243N 14 254 F200M 1456 F200Y 14 80 F200M/T243N 14 168 S244K 14 8

The T243N was the best mutant of all tested for activity in theproduction of R,R monatin.

Example 27 Stabilization of the T243N Mutant of the D-Aminotransferasefrom Strain ATCC 4978

As shown in Example 25, the initial activity of the T243N mutant DAT issignificantly higher than the B. sphaericus DAT, but activity decreasesmore rapidly. Additional experiments, using the anaerobic protocoldescribed below, indicated that the initial activity of the T243N mutantDAT was up to 8-fold higher than the B. sphaericus DAT, however theactivity decreased rapidly even under the anaerobic conditions. Thefollowing studies were done to try to maintain the higher activity foran extended period of time.

The T243N mutant of the D-aminotransferase from strain 4978 (describedin Example 25) and the S. meliloti HMG aldolase were purified as theHIS₆-tagged proteins as described below. The SEQ ID NO:276 aldolase waspurified as described in Example 23.

Purification of the DAT from ATCC 4978 (T243N Mutant)

The T243N mutant of the D-aminotransferase from ATCC strain 4978 with anamino-terminal HIS₆-purification tag (described in Example 26) wasproduced using the EMD Biosciences Overnight Express System II(solutions 1-6) (Novagen, Madison, Wis.) containing 50 μg/mL kanamycinin shake flasks. This expression system induces the expression ofIPTG-inducible systems without the need to monitor cell growth. Afterinoculation of 200-mL aliquots of the medium (in 1 L flasks) from eitherliquid cultures or plates of the E. coli BL21(DE3) host carrying thegene for the T243N mutant D-aminotransferase from ATCC strain 4978 onthe plasmid pET28b, the cultures were incubated at 30° C. overnight withshaking at 225 rpm. When the OD600 had reached a minimum of 6, the cellswere harvested by centrifugation and washed once with buffer.

Cell free extract was prepared using EMD Biosciences BugBuster® (primaryamine-free) Extraction Reagent (Novagen, Madison, Wis.) containing 1μL/mL Benzonase® Nuclease (Novagen, Madison, Wis.), 5 μL/mL ProteaseInhibitor Cocktail Set II (Calbiochem—Novabiochem Corp., San Diego,Calif.), and 0.033 μL/mL rLysozyme™ (Novagen, Madison, Wis.) followingthe manufacturer's protocol. All subsequent purification steps werecarried out at 4° C. The cell extract was centrifuged for 20-30 minutesat 15,000×g to remove the cell debris. A 20-25 mL aliquot of the cellfree extract was applied to a 45 mL column of GE Healthcare ChelatingSepharose™ Fast Flow resin (nickel (II) form) (GE Healthcare,Piscataway, N.J.) that had been previously equilibrated with 100 mMpotassium phosphate containing 200 mM sodium chloride and 50 μM PLP. Togenerate the nickel form of the resin, the resin was washed with 150 mLof 200 mM nickel (II) sulfate hexahydrate and then with 150 mL ofdistilled water. After loading the sample, the column was washed/elutedwith 150 mL of the equilibration buffer containing 25 mM imidazole, 150mL of the equilibration buffer containing 50 mM imidazole and 150 mL ofthe equilibration buffer containing 500 mM imidazole. The HIS₆-taggedprotein eluted in the last wash. The 500 mM imidazole wash wasconcentrated with Millipore/Amicon Centricon Plus-70 centrifugal filterdevices (MWCO 10 kDa) (Millipore, Billerica, Mass.) to 15-20 mLaccording to the manufacturer's instructions. The imidazole and sodiumchloride were removed by passage through disposable GE Healthcare PD10columns (GE Healthcare, Piscataway, N.J.) (2.5 mL sample per column)previously equilibrated with 100 mM potassium phosphate, pH 7.8containing 0.5 μM PLP. The purified aminotransferase was eluted with 3.5mL per column of the same buffer. The protein concentration of eachfraction was determined using the Pierce BCA assay kit (Pierce,Rockford, Ill.) with BSA as the protein standard.

The purity of each fraction and the level of expression in the cell freeextract fraction were determined using a Bio-Rad Experion microcapillarychip system (Bio-Rad, Hercules, Calif.) or using Bio-Rad 4-15%SDS-polyacrylamide gradient gels (Bio-Rad, Hercules, Calif.) run in aMini PROTEAN® 3 cell apparatus. The protein was visualized in thepolyacrylamide gels using Bio-Rad Bio-Safe G-250 Coomassie stain(Bio-Rad, Hercules, Calif.) and destained with water. Typically thisprocedure produces˜20 mg of enzyme from 200 mL of overnight culture thatis 85-90% pure as judged by the Experion software or from analysis ofthe SDS-PAGE gels. Aliquots (1-5 mL) of the purified enzyme were storedat −80° C. until use.

The purification of the SEQ ID NO:276 aldolase is as described inExample 23.

Purification of the S. meliloti HMG Aldolase

The S. meliloti HMG aldolase with an amino-terminal HIS₆-purificationtag (cloning described in U.S. Published Application No. 20040063175 andWO 03091396 A2) was produced by induction of cultures grown inLuria-Bertani broth containing 50 mg/L kanamycin with 0.2 mM IPTG. Afterinoculation of 800-mL aliquots of the medium from either liquid culturesor plates of the E. coli BL21(DE3) host carrying the gene for the S.meliloti HMG aldolase in pET30(Xa/LIC), the cultures were incubated at37° C. with shaking at 225 rpm. When the optical density reached anOD_(600nm) of 0.5-0.75, the IPTG was added and the cultures wereincubated 30° C. with shaking at 225 rpm for 4 hours. The cells wereharvested by centrifugation and washed once with buffer.

To prepare cell free extract containing the S. meliloti aldolase, thecells were suspended in 3-4 volumes of 50 mMEPPS(N-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid), pH 8.2containing 100 mM NaCl and then disrupted using a Microfluidicshomogenizer (Microfluidics, Newton, Mass.) (3 passes at 18,000 psi),maintaining the temperature of the suspension at less than 15° C. Thecell suspension was centrifuged for 20-30 minutes at 15,000-20,000×g toremove the cell debris. The HIS₆-tagged protein was purified using EMDBiosciences HIS-Bind® columns (Novagen, Madison, Wis.) following themanufacturer's recommended protocol with one exception: the columns werewashed with a 1:1 solution of Binding buffer:Wash buffer instead of theWash buffer alone. The elution fraction was concentrated withMillipore/Amicon 15 mL centrifugal filter devices (MWCO 5 kDa)(Millipore, Billerica, Mass.) to 7-10 mL according to the manufacturer'sinstructions. The imidazole and sodium chloride were removed by passagethrough disposable GE Healthcare PD10 columns (GE Healthcare,Piscataway, N.J.) (2.5 mL sample per column) previously equilibratedwith 50 mM EPPS, pH 8.2 containing 100 mM NaCl. The purified aldolasewas eluted with 3.5 mL per column of the same buffer. The proteinconcentration of each fraction was determined using the Pierce BCA assaykit (Pierce, Rockford, Ill.) using BSA as the protein standard.

The purity of each fraction and the level of expression in the cell freeextract fraction were determined using Bio-Rad 4-15% SDS-polyacrylamidegradient gels (Bio-Rad, Hercules, Calif.) run in a Mini PROTEAN® 3 cellapparatus. The protein was visualized in the polyacrylamide gels usingBio-Rad Bio-Safe G-250 Coomassie stain (Bio-Rad, Hercules, Calif.) anddestained with water. Typically, this procedure produces ˜15-20 mg ofenzyme from 800 mL of culture and is 85-95% pure. Aliquots (1-5 mL) ofthe purified enzyme were stored at −80° C. until use.

Monatin Production Assays

Conical polypropylene tubes (14 mL) containing 143 mg of D-tryptophanwere de-oxygenated in an anaerobic glove box overnight. Stock solutionsof 1M EPPS buffer (pH 8.2), 2M MgCl₂, 2M sodium pyruvate and 10 mM PLPwere prepared in degassed water and equilibrated in an anaerobic glovebox overnight. Stock solutions of 10% (v/v) Tween 80, 1% (v/v) Tween 20,1% (v/v) Triton X-100, 100% acetone, 100% ethanol and 50% (w/v) glycerolwere equilibrated in the anaerobic glove box along with 0.7 g each oftrehalose, inositol, sorbitol and erythritol in 2 mL microcentrifugetubes. Preparations of the purified enzymes were thawed on ice and usedimmediately in the anaerobic glove box. The stock solutions were addedto the 14 mL conical tubes to give a final concentration of 100 mM EPPS,200 mM pyruvate, 100 mM tryptophan, 1 mM MgCl₂, 0.05 mM PLP, 0.5 mg/mLD-aminotransferase, and 0.01 mg/mL of SEQ ID NO:276 aldolase or 0.05mg/mL of S. meliloti HMG aldolase. The proposed enzyme stabilizingcomponents were added at various final concentrations (Tables 38 and 39)to bring the final reaction volume to 7 mL per tube. The reactions wereincubated at room temperature in the anaerobic glove box with gentleagitation for up to 24 hours. Samples were removed periodically andanalyzed for monatin as described in Example 1 using the LC/MS/MSmultiple reaction monitoring method. The initial rates were calculatedfrom the samples withdrawn between 0 and 3 h after the addition of theenzyme.

TABLE 38 Fold Improvement Fold Improvement in in Initial Rate of FinalAdditive Monatin Formation Monatin Titer (20 h) None 1.0 1.0 0.01% (v/v)Tween 80 1.3 1.4 0.1% (v/v) Tween 80 1.3 1.5 0.01% (v/v) Tween 20 1.11.5 0.01% (v/v) Triton X-100 1.1 1.2 5% (v/v) Acetone 0.4 0.3 5% (v/v)Ethanol 0.7 0.5 1% (w/v) Glycerol 1.9 1.1 5% (w/v) Glycerol 1.4 1.4 10%(w/v) Glycerol 1.1 1.7 10% (w/v) Trehalose 1.0 1.3 10% (w/v) Inositol1.3 1.5 10% (w/v) Sorbitol 1.1 1.3 10% (w/v) Erythritol 0.8 1.0

TABLE 39 (SEQ ID NO: 276 aldolase) Fold Improvement in Fold Improvementin Initial Rate Final Additive of Monatin Formation Monatin Titer at 22h 0.01% (v/v) Tween 80 1.0 1.0 1% (w/v) Glycerol 1.2 0.9 5% (w/v)Glycerol 1.5 1.5 10% (w/v) Glycerol 1.7 2.1

The addition of 0.01%-0.1% (v/v) detergent, such as Triton X-100, Tween20 or Tween 80, or 1%-10% (w/v) polyol, such as glycerol, trehalose,inositol or sorbitol, improved the stability of the T243ND-aminotransferase over the lifetime of the experiment.

Example 28 A: Cloning of Pseudomonas putida KT2440 Broad-SpecificityAmino acid Racemase (BAR)

A BAR was identified in P. putida KT2440 using information fromliterature (Roise, D., Soda, K., Yagi, T., Walsch, C. T., Biochemistry23, 5195-5201, (1984)). The active site of a BAR enzyme from P. striatawas sequenced and reported-LTAVLKADAYGXGIGL (SEQ ID NO:375). Thissequence was used to BLAST the P. putida KT2440 genome sequenceavailable in NCBI. A protein with a nearly identical consensus sequencewas identified. Primers were designed to clone the gene from genomic DNAobtained from the American Type Culture Collection (ATCC, Manassas, Va.)

(5′-AGAAGACATATGCCCTTTCGCCGTAGGG-3′ (SEQ ID NO: 376 and5′-AGAAGAGGATCCTCAGTCGACGAGTATCTTCG-3′) (SEQ ID NO: 377)).

PCR was conducted under standard conditions and the PCR product waspurified (QIAquick PCR purification kit, Qiagen, Valencia, Calif.). Thepurified PCR product was digested with Nde I and BamH I. The digestedPCR product was gel purified (QIAquick Gel Extraction Kit, Qiagen,Valencia, Calif.) and ligated to pET30 and pET28 that had been digestedand gel purified in a similar manner. Clones with inserts were sequenced(Agencourt, Beverly, Mass.) and isolates with the correct sequence wereidentified (pET30 KT2440 BAR and pET28 KT2440BAR) and used in laterstudies.

The KT2440 BAR DNA sequence is: (SEQ ID NO: 378)atgccctttcgccgtacccttctggctgcatccctggcacttctgatcaccggacaggcccccctgtatgcggcaccaccgttgtcgatggacaacggcaccaacaccctgaccgtgcaaaacagcaatgcctgggtcgaagtcagcgccagcgccctgcagcacaacatccgcacgctgcaggccgagctggccggcaagtccaagctgtgcgccgtgctcaaggccgatgcctatggccacggtatcggcctggtaatgccatcgatcatcgcccaaggcgtgccctgcgtggcggtggccagcaacgaggaggcccgcgtggtccgcgccagtggcttcaccgggcaactggtgcgggtacgcctggccagcctcagcgagctggaagatggcttgcagtacgacatggaagagctggtgggcagcgcggaatttgcccgccaggccgatgccatcgccgcgcgccatggcaagaccttgcgcattcacatggcgctcaactccagcggcatgagccgcaacggggtggagatggccacctggtccggccgtggcgaagcgctgcagatcaccgaccagaagcacctcaagctggtcgcgctgatgacccacttcgccgtggaagacaaggacgatgtacgcaagggcctggcggcattcaacgagcagaccgactggttgatcaagcacgccaggctggaccgcagcaagctcaccctgcacgccgccaactcgttcgctacgctggaagtgccggaagcgcgcctggacatggtacgaacgggtggcgcgctgttcggcgacaccgtgccggcgcgcaccgagtacaaacgtgcgatgcagttcaaatcgcacgtggcggcggtgcacagctatccggccggcaacaccgtgggctatgaccgcaccttcaccctggcccgtgattcgcggctggccaacattacggtcgggtactccgatggctaccgccgggtattcaccaacaagggccatgtgctgatcaacggccaccgtgtgccggtcgtgggcaaggtgtcgatgaacacgctgatggtcgatgtcaccgacttccctgatgtgaaggggggtaacgaagtggtgctgttcggcaagcaggccgggggcgaaatcacccaggccgagatggaagaaatcaacggcgcgttgctcgccgatttgtacaccgtatggggcaattccaacccgaagatactcgtcgactga.The KT2440 BAR amino acid sequence is: (SEQ ID NO: 379)MpfrrtllaaslallitgqaplyaapplsmdngtntltvqnsnawvevsasalqhnirtlqaelagksklcavlkadayghgiglvmpsiiaqgvpcvavasneearvvrasgftgqlvrvrlaslseledglqydmeelvgsaefarqadaiaarhgktlrihmalnssgmsrngvematwsgrgealqitdqkhlklvalmthfavedkddvrkglaafneqtdwlikharldrskltlhaansfatlevpearldmvrtggalfgdtvparteykramqfkshvaavhsypagntvgydrtftlardsrlanitvgysdgyrrvftnkghvlinghrvpvvgkvsmntlmvdvtdfpdvkggnevvlfgkqaggeitqaemeeingalladlytvwgnsnpkilvdB) Purification of P. putida KT2440 BAR.

The pET30 KT2440 BAR plasmid described above was transformed into BL21DE3 pLysS (Invitrogen, Carlsbad, Calif.). The resulting strain was grownin LB or Terrific Broth at 37° C. with aeration to an OD_(600nm) of0.4-0.6 and induced with 1 mM IPTG. Incubation was continued 3-4 hoursat 37° C. with aeration. The cells were harvested by centrifugation andthe cell pellet was stored at −80° C. until use. The cell pellet wasthawed on ice. The cells were lysed with BugBuster (Novagen, Madison,Wis.) and Benzonase (Novagen, Madison, Wis.). Cell debris was removed bycentrifugation and the cell free extract was either used immediately orstored at −80° C. The KT2440 BAR gene was also cloned into theNdeI-BamHI sites of pET28 and transformed into BL21 DE3 pLysS. Thisconstruct did not appear to express soluble protein very efficiently sothe untagged version (pET30 KT2440 BAR) was used in future studies.

The extract was applied to an UnoQ column (Bio-Rad, Hercules, Calif.)that had been equilibrated with at least 5 column volumes buffer A (25mM potassium phosphate pH 8.0, 10 μM pyridoxal-5′-phosphate (PLP)). Thecolumn was washed with 2 column volumes of buffer A. The protein waseluted with a linear gradient of buffer B (buffer A+1M NaCl) from 0-100%buffer B over 20 column volumes and 5 ml fractions were collected fromthe time the gradient started. Fractions were assayed using the AmplexRed method described in Example 4-part 7. Briefly, 100 μg D-amino acidoxidase (Sigma-Aldrich, St. Louis, Mo.), 0.05 mM FAD, 25 mM L-trp, and asmall volume of the fraction to be assayed were combined in 50 μL H₂Oand added to 50 μL Amplex Red reaction buffer prepared as directed inthe manufacturer's protocol. Fractions with activity were desalted witha PD-10 column (GE Healthcare, Piscataway, N.J.) and concentrated withAmicon centrifugal concentrators (Millipore, Billercia, Mass.). Purifiedprotein was stored at −80° C.

C) Production and Assay of an Alanine Racemase Mutant Y354A ofGeobacillus stearothermophilus with Tryptophan Racemase Activity

The wild-type Geobacillus stearothermophilus alanine racemase (SEQ IDNO:380, shown below was cloned into pET30 was used as a template forsite-directed mutagenesis to make the Y354A change. The gene of SEQ IDNO:380 can be amplified by standard PCR protocols and cloned usingstandard recombinant DNA techniques. The gene of SEQ ID NO:380 can alsobe reconstructed by any method known to a person of ordinary skill inthe art, such as assembly PCR methods known to one skilled in the art.

The wild-type Geobacillus stearothermophilus alanine racemase DNA andamino acid sequences is shown below as SEQ ID NO:380:

(SEQ ID NO: 380) atggacgagt ttcaccgcga tacgtgggcg gaagtggatt tggacgccatttacgacaat gtggagaatt tgcgccgttt gctgccggac gacacgcaca ttatggcggtcgtgaaggcg aacgcctatg gacatgggga tgtgcaggtg gcaaggacag cgctcgaagcgggggcctcc cgcctggcgg ttgccttttt ggatgaggcg ctcgctttaa gggaaaaaggaatcgaagcg ccgattctag ttctcggggc ttcccgtcca gctgatgcgg cgctggccgcccagcagcgc attgccctga ccgtgttccg ctccgactgg ttggaagaag cgtccgccctttacagcggc ccttttccta ttcatttcca tttgaaaatg gacaccggca tgggacggcttggagtgaaa gacgaggaag agacgaaacg aatcgtagcg ctgattgagc gccatccgcattttgtgctt gaaggggtgt acacgcattt tgcgactgcg gatgaggtga acaccgattatttttcctat cagtataccc gttttttgca catgctcgaa tggctgccgt cgcgcccgccgctcgtccat tgcgccaaca gcgcagcgtc gctccgtttc cctgaccgga cgttcaatatggtccgcttc ggcattgcca tgtatgggct tgccccgtcg cccggcatca agccgctgctgccgtatcca ttaaaagaag cattttcgct ccatagccgc ctcgtacacg tcaaaaaactgcaaccaggc gaaaaggtga gctatggtgc gacgtacact gcgcagacgg aggagtggatcgggacgatt ccgatcggct atgcggacgg ctggctccgc cgcctgcagc actttcatgtccttgttgac ggacaaaagg cgccgattgt cggccgcatt tgcatggacc agtgcatgatccgcctgcct ggtccgctgc cggtcggcac gaaggtgaca ctgattggtc gccaaggggacgaggtaatt tccattgatg atgtcgctcg ccatttggaa acgatcaact acgaagtgccttgcacgatc agttatcgag tgccccgtat ttttttccgc cataagcgta taatggaagtgagaaacgcc gttggccgcg ga.

The encoded amino acid sequence of the Alanine Racemase (Geobacillusstearothermophilus) is shown below as SEQ ID NO:381:

(SEQ ID NO: 381) Met Asp Glu Phe His Arg Asp Thr Trp Ala Glu Val Asp LeuAsp Ala Ile Tyr Asp Asn Val Glu Asn Leu Arg Arg Leu Leu Pro Asp Asp ThrHis Ile Met Ala Val Val Lys Ala Asn Ala Tyr Gly His Gly Asp Val Gln ValAla Arg Thr Ala Leu Glu Ala Gly Ala Ser Arg Leu Ala Val Ala Phe Leu AspGlu Ala Leu Ala Leu Arg Glu Lys Gly Ile Glu Ala Pro Ile Leu Val Leu GlyAla Ser Arg Pro Ala Asp Ala Ala Leu Ala Ala Gln Gln Arg Ile Ala Leu ThrVal Phe Arg Ser Asp Trp Leu Glu Glu Ala Ser Ala Leu Tyr Ser Gly Pro PhePro Ile His Phe His Leu Lys Met Asp Thr Gly Met Gly Arg Leu Gly Val LysAsp Glu Glu Glu Thr Lys Arg Ile Val Ala Leu Ile Glu Arg His Pro His PheVal Leu Glu Gly Val Tyr Thr His Phe Ala Thr Ala Asp Glu Val Asn Thr AspTyr Phe Ser Tyr Gln Tyr Thr Arg Phe Leu His Met Leu Glu Trp Leu Pro SerArg Pro Pro Leu Val His Cys Ala Asn Ser Ala Ala Ser Leu Arg Phe Pro AspArg Thr Phe Asn Met Val Arg Phe Gly Ile Ala Met Tyr Gly Leu Ala Pro SerPro Gly Ile Lys Pro Leu Leu Pro Tyr Pro Leu Lys Glu Ala Phe Ser Leu HisSer Arg Leu Val His Val Lys Lys Leu Gln Pro Gly Glu Lys Val Ser Tyr GlyAla Thr Tyr Thr Ala Gln Thr Glu Glu Trp Ile Gly Thr Ile Pro Ile Gly TyrAla Asp Gly Trp Leu Arg Arg Leu Gln His Phe His Val Leu Val Asp Gly GlnLys Ala Pro Ile Val Gly Arg Ile Cys Met Asp Gln Cys Met Ile Arg Leu ProGly Pro Leu Pro Val Gly Thr Lys Val Thr Leu Ile Gly Arg Gln Gly Asp GluVal Ile Ser Ile Asp Asp Val Ala Arg His Leu Glu Thr Ile Asn Tyr Glu ValPro Cys Thr Ile Ser Tyr Arg Val Pro Arg Ile Phe Phe Arg His Lys Arg IleMet Glu Val Arg Asn Ala Val Gly Arg Gly

The mutagenesis was performed using the QuickChange-Multi site-directedmutagenesis kit (Stratagene, La Jolla, Calif.). The following mutagenicprimer was used to make the Y354A change,5′-gccatttggaaacgatcaacgcggaagtgccttgcacgatcag-3′ (SEQ ID NO:382). Thesite-directed mutagenesis was done as described in the manufacturer'sprotocol. Several isolates were sequenced (Agencourt, Beverly, Mass.)and an isolate with the correct sequence was selected and used forfurther analysis.

The pET30Y354A single mutant was transformed into E. coliBL21(DE3)pLysS. Purified protein was prepared in the following manner.The strain was grown in LB or Terrific Broth (at 37° C. with aeration)to an OD_(600m) of 0.4-0.6 and induced with 1 mM IPTG. Incubation wascontinued at 37° C. with aeration for ˜3 hours. The cells were harvestedby centrifugation and the cell pellet was stored at −80° C.

The cell pellet was thawed on ice and then re-suspended in anappropriate volume of BugBuster (Novagen, Madison, Wis.) plus Benzonasenuclease (Novagen, Madison, Wis.). Cell debris was removed bycentrifugation, and the cell-free extract was applied to a HIS-Bindcolumn (Novagen, Madison, Wis.) that had been equilibrated with Bindingbuffer. The column was washed with Binding buffer and Wash buffer andthe protein was eluted with Elution buffer (as directed in themanufacturer's protocol). The purified protein was desalted using aPD-10 column (GE Healthcare, Piscataway, N.J.). The protein was desaltedinto 50 mM potassium phosphate pH 8.0 and 10 μM pyridoxal-5′-phosphateaccording to the manufacturer's protocol. The protein was concentratedusing an Amicon centrifugal concentrator (Millipore, Billercia, Mass.).The purified and concentrated protein was divided into small aliquotsand stored at −80° C. until use.

The purified Y354A was compared to wild-type alanine racemase (preparedin the manner described above) in both alanine and tryptophan assays.Assays were performed at 37° C. in 50 mM potassium phosphate buffer, pH8, and 10 μM PLP using 400 μL of concentrated purified protein (>1mg/ml) and 50 mM substrate. Detection of D-alanine and D-tryptophan wasperformed using the chiral amino acid methodology described in Example1.

TABLE 40 D isomer produced Enzyme Substrate Time (ppm) Wild-typeL-tryptophan 0 nd* 10 nd 30 nd 60 nd 1080 nd Y354A 0 nd 10 198 30 568 601386 1080 10080 Wild-type L-alanine 0 5140 10 5960 30 6280 60 6500 10805040 Y354A 0 4760 10 4980 30 4980 60 4200 1080 5000 *nd = none detected

These data were analyzed without the use of an internal standard; thus,these data are semi-quantitative and should be used for comparativepurposes. Nonetheless, these results show that the Y354A single mutationis sufficient to broaden the specificity of the alanine racemase so thatit can catalyze amino acid racemization using alternative substrates.

D) Assay of the BAR Enzyme.

The BAR enzyme was assayed as follows. Amplex Red assays were set up asdescribed in this example. P. putida KT2440 BAR was used at 200 μg(purified as described in this example). Wildtype G. stearothermophilusalanine racemase and the Y354A were purified as described in thisexample and used at either 200 μg/mL or 1000 μg/mL. CE is cell-freeextract that was prepared as described in this example. The results forthe 60 minute time point are shown in the Table 41 below.

TABLE 41 Fluorometer reading Enzyme (at 60 minutes) BAR (200) 56943Y354A (200) 7860 Y354A (1000) 13587 WT alanine racemase (200) 3646 WTalanine racemase (1000) 3639 BAR CE (5 μl) 16228 BAR CE (10 μl) 26662BAR CE (50 μl) >58000 No Enzyme 1510

The purified protein was also assayed for tryptophan racemase activityin 50 mM potassium phosphate pH 8, 10 μM PLP, and 30 mM L-tryptophan asdescribed above. Either 200 μg/mL or 1000 μg/mL of purified enzyme wasused in the assays (indicated in parentheses). D-tryptophan was analyzedusing the chiral amino acid method described in Example 1 for detection.

TABLE 42 Enzyme Time D-tryptophan (ppm) BAR (200) 0 0 5 172 10 410 20844 30 1318 60 2362 120 2594 240 2762 1080 2294 Y354A (200) 0 0 5 0 10 020 0 30 12 60 22 120 44 240 56 1080 368 Y354A (1000) 0 0 5 0 10 12 20 1830 40 60 80 120 146 240 218 1080 1164

The assays indicate that the P. putida KT2440 BAR enzyme is much moreactive on tryptophan than the G. stearothermophilus derived enzyme andthe Y354A mutant thereof.

E) Monatin Production with P. putida KT2440 BAR.

A monatin production assay was done with the purified P. putida KT2440BAR (as purified above) (100 μg) or purified Y354A (as purified above)(500 μg), D-aminotransferase (BioCatalytics AT-103 (Pasadena, Calif.))(500 μg), and the aldolase of SEQ ID NO:276) (as purified in Example 23)(50 μg). The monatin production experiment starting with L-tryptophanwas done as follows. In addition to the enzymes above, the followingwere added per 1 mL of reaction mixture: 4 mM MgCl₂, 50 mM L-tryptophan,100 mM sodium pyruvate, 100 mM potassium phosphate buffer pH 7.5, and0.05 mM PLP.

As a control, the experiment was done as described above withoutracemase and starting with D-tryptophan instead of L-tryptophan. Asummary of the results is presented in Table 43 below.

TABLE 43 Sub- Race- Total % % % % strate mase Time Monatin R,R S,S R,SS,R L-Trp Y354A 2 hours None detected 18 hours None detected L-trp BAR 2hours None detected 18 hours 38.6 ppm 92.1 5 2.9 L-trp None 2 hours Nonedetected 18 hours None detected D-trp None 2 hours 19.9 ppm Not Not NotNot tested tested tested tested 18 hours 221.25 ppm  97.8 0.2 2

No monatin was detected using Y354A in this experiment. This racemasehas been used in the past (data not shown) to produce monatin, but amuch higher level of enzyme was used (at least 2 mg and up to 10 mg tosee higher levels of monatin). The P. putida KT2440 BAR was used toproduce monatin from L-tryptophan. The 100 μg used in this experimentwas not enough to see monatin production after two hours but was enoughto see monatin production after 18 hours. The stereoisomer distributionindicated that most of the monatin produced is the R,R isomer. There wasno R,S isomer produced. This result indicates that KT2440 BAR is notable to detectably racemize the R,R isomer of monatin (racemization ofthe R,R isomer would produce the R,S isomer). There was a significantamount of the S,S isomer produced in this experiment. This is probablydue to the fact that the AT-103 used in this experiment is not highlypurified and may contain L-aminotransferases from the cellular extract,and that there is a large amount of L-tryptophan present to serve as anamino donor for transamination of S-MP.

Example 29 Cloning and Expression of Pseudomonas taetrolens ArginineRacemase Experimental Overview

Pseudomonas taetrolens (also known as P. graveolens) arginine racemase(Genbank Accession No. AB096176, nucleic acid sequence) and an 1384Mmutant thereof, was cloned, expressed, and tested for activity inconversion of L-tryptophan to D-tryptophan. This gene is 72% identicalto the P. putida BAR gene from KT2440 and 73% identical to the P. putidaBAR gene from NBRC 12996 described above. The amino acid sequence is 72%identical to both P. putida BAR proteins.

Polymerase Chain Reaction Protocol

Pseudomonas taetrolens (ATCC 4683) was grown in nutrient broth at 28° C.with shaking at 225 rpm. Polymerase chain reaction was performed onwhole cells using primers designed with 5′ restriction sites andoverhangs for cloning into the pET 28 and pET30 vectors (Novagen,Madison, Wis.).

The primer sequences were: N term: (SEQ ID NO: 408)5′-ATAATACATATGCCCTTCTCCCGTACCC-3′ and C term: (SEQ ID NO: 409)5′-GCGGCGGGATCCTTACTGATCTTTCAGGATT-3′.

The gene derived from P. taetrolens was amplified using the followingPCR protocol. Twenty-five μL of grown cells were lysed at 96° C. for 10minutes. Cell debris was removed by centrifugation and the supernatantwas used as template for PCR. A 100 μL reaction contained 5 μL template(lysed cell supernatant), 1.6 μM of each primer, 0.3 mM each dNTP, 10 UrT^(th) Polymerase XL (Applied Biosystems, Foster City, Calif.), 1×XLbuffer and 1 mM Mg(OAc)₂. The thermocycler program used included a hotstart at 94° C. for 3 minutes, 8 repetitions of the following steps: 94°C. for 30 seconds, 52° C. for 30 seconds, and 68° C. for 2 minutes,followed by 22 repetitions of the following steps: 94° C. for 30seconds, 58° C. for 30 seconds, and 68° C. for 2 minutes. After the 22repetitions, the sample was maintained at 68° C. for 7 minutes and thenstored at 4° C. This PCR protocol produced a product of 1230 bp.

Cloning

The PCR product was gel purified from 0.8% TAE-agarose gel using theQiagen gel extraction kit (Qiagen, Valencia, Calif.). The product wasTOPO cloned and transformed into TOP 10 cells according tomanufacturer's protocol (Invitrogen, Carlsbad, Calif.). Plasmid DNA waspurified from the resulting transformants using the Qiagen spin miniprepkit (Qiagen, Valencia, Calif.) and screened for the correct inserts byrestriction digest with Nde I and BamH I. The sequences of plasmidsappearing to have the correct insert were verified by dideoxy chaintermination DNA sequencing with universal M13 forward and M13 Reverseprimers.

The correct TOPO clone was digested with restriction enzymes Nde I andBamH I following the manufacturer's recommended protocols (New EnglandBiolabs, Beverly, Mass.) and gel purified from 0.8% TAE-agarose gelsusing the Qiagen gel extraction kit (Qiagen, Valencia, Calif.). VectorspET 28 and pET 30 were prepared by digestion with restriction enzymesNde I and BamH I followed by treatment with shrimp alkaline phosphatase(Roche, Indianapolis, Ind.) and purification from 0.8% TAE-agarose gelsusing the Qiagen gel extraction kit (Qiagen, Valencia, Calif.). Thedigested vectors and insert were ligated using the Rapid™ DNA LigationKit (Roche, Indianapolis, Ind.). Approximately 50 ng of treated insert,100 ng of treated vector (3 to 1 molar ratio of insert to vector), 5 Uof T4 DNA ligase, and 1× ligation buffer were incubated for 5 minutes atroom temperature. The ligation reaction was desalted using the High PurePCR Product Purification Kit (Roche, Indianapolis, Ind.) and used totransform E. coli DH10B electrocompetent cells (Invitrogen, Carlsbad,Calif.). Ten μL of each ligation reaction was added to 40 μL of DH10Bcells, which were transformed by electroporation using the BioRad GenePulsar II under the following conditions: 2.5 kV, 25 μF, 200 ohm in a0.2 cm cuvette. The cells were allowed to recover in 1 mL of roomtemperature SOC for 1 hour at 37° C. with shaking at 225 rpm. Cells wereplated on LB plates containing kanamycin (50 μg/mL). Plasmid DNA waspurified from the resulting transformants using the Qiagen spin miniprepkit (Qiagen, Valencia, Calif.) and screened for the correct inserts byrestriction digest with Nde I and BamH I.

Gene Expression and Assays

Plasmid DNA was transformed into E. coli expression host BL21(DE3) pLysS(Novagen, Madison, Wis.). The cultures were grown and the plasmids wereisolated using the Qiagen miniprep kit (Qiagen, Valencia, Calif.) andanalyzed by restriction digest to confirm identity.

Induction in BL21DE3 pLysS was initially performed in both pET 28(histidine-tagged) and pET 30 (untagged) vectors. A time course studywas performed with cultures grown at 37° C. in 100 mL LB containingkanamycin (50 mg/L) to an OD₆₀₀ of 0.5 and induced with 100 μM IPTG(isopropyl thiogalacatoside) and sampled at 0 and 3 hours postinduction. Cells from 0 hour and 3 hour time points were resuspended in1× sodium dodecyl sulfate buffer containing 2-mercaptoethanol and heatedat 95° C. for 10 minutes, and cooled. Aliquots of these total cellularprotein samples were analyzed by SDS-PAGE using a 4-15% gradient gel.

Cell extracts were also prepared from the 3 hour cultures by suspendingcell pellets from 5 mL of culture in Novagen BugBuster™ reagentcontaining benzonase nuclease and protease inhibitor cocktail set #3(Calbiochem-Novabiochem Corp., San Diego, Calif.) at room temperaturefor 20 minutes with gentle shaking and centrifuging at 16,000×g toremove cell debris. The supernatants (cell extracts) were loaded onto4-15% gradient gels for analysis of the cellular soluble proteins.

The 3 hour sample from cloned P. taetrolens arginine racemase showed atotal protein band that corresponded to the correct size (approximately45 kDa) in the pET 30 (untagged) vector. The P. taetrolens pET 30 geneproduct was over-expressed at a higher level than the P. taetrolens pET28 (histidine-tagged) gene product, but neither of the vectors gave avisible soluble protein band.

Cells from the induced cultures (100 mL) were centrifuged and washedonce with 0.85% NaCl. Cell pellets were resuspended in 5 mL/g wet cellweight of BugBuster™ (Novagen, Madison, Wis.) reagent containing 5 μL/mLprotease inhibitor cocktail set #3 (Calbiochem-Novabiochem Corp., SanDiego, Calif.) and 1 μL/mL benzonase nuclease. Samples were incubated atroom temperature for 20 minutes on an orbital shaker. Insoluble celldebris was removed by centrifugation at 16,000×g for 20 minutes at 4° C.

Cell extracts were assayed for tryptophan racemase activity using thefollowing protocol. One mL reactions were carried out in 50 mM potassiumphosphate (pH 8.0), 0.05 mM PLP and 30 mM L tryptophan. The reactionswere initiated by the addition of cell free extracts and were incubatedat 30° C. overnight. Sample aliquots were taken after overnightincubation (zero minute samples served as control reaction).Concentrated formic acid (5 μL) was added to each 250 μL sample aliquotto stop the reaction and the precipitated protein was removed bycentrifugation. Supernatants were removed and frozen at −80° C. untilthey were analyzed for D-tryptophan by the chiral amino acid methoddescribed in Example 1.

Assay results from cell extracts from pET28 and pET30 induction with 100μM IPTG (3 hours) demonstrate that P. taetrolens clones show racemaseactivity on L-tryptophan. Again, the tagged version of the BAR appearsto be less active and may precipitate or be less soluble than theuntagged (pET28). Table 44, below, shows the initial results, althoughnot quantitative as very poor soluble protein was obtained.

TABLE 44 D-trp Time Racemase extract conc Treatment Point Substrate (200μg) (μg/mL) pET28/P. taetrolens 0 L-trp 500 μL nd pET30/P. taetrolens 0L-trp 500 μL nd pET28/P. taetrolens overnight L-trp 500 μL 140 pET30/P.taetrolens overnight L-trp 500 μL 226

Induction of the pET30 (untagged) construct was repeated using sameconditions as mentioned above and a visible soluble protein band wasobserved in SDS-PAGE. The assay was repeated using same the conditionsdescribed above and the results, as shown in Table 45 below, wereobtained.

TABLE 45 Racemase extract D-trp conc Treatment Time Point Substrate (μL)(μg/mL) P. taetrolens-pET30 0 L-trp 300 Nd P. taetrolens-pET30 0 L-trp150 Nd P. taetrolens-pET30 2 hr L-trp 300 319 P. taetrolens-pET30 2 hrL-trp 150 308 P. taetrolens-pET30 Overnight L-trp 300 1586 P.taetrolens-pET30 Overnight L-trp 150 1658

Again, it was noted that doubling of the volumes did not scale to moreactivity. For future work, it was determined to remove the protein fromBugbuster as quickly as possible after preparation of cell extracts andto store the protein in 50 mM phosphate buffer pH 8 containing 0.01 mMPLP. The detergent in Bugbuster may inhibit the reaction or may cause aloss of activity upon storage.

Induction of the pET30 construct was carried out again and the cellextract was processed with anion exchange chromatography (as in Example28) to give a more pure extract. The assay was repeated with thispartially purified prep. The numbers in parenthesis in the Racemaseextract column of Table 46 below indicate the approximate amount ofpartially purified racemase enzyme used in the assay. The results of theassay are shown in Table 46 below.

TABLE 46 D-trp conc Enzyme Source Time Point Substrate Racemase extract(μg/mL) KT2440 0 L-trp  75 μL (90 μg) nd NBRC 12996 0 L-trp  42 μL (200μg) nd NBRC 12996 0 L-trp  21 μL (100 μg) nd P. taetrolens 0 L-trp 108μL (200 μg) nd P. taetrolens 0 L-trp  54 μL (100 μg) nd KT2440 2 hrL-trp  75 μL (90 μg) 661 NBRC 12996 2 hr L-trp  42 μL (200 μg) 408 NBRC12996 2 hr L-trp  21 μL (100 μg) 208 P. taetrolens 2 hr L-trp 108 μL(200 μg) 862 P. taetrolens 2 hr L-trp  54 μL (100 μg) 547 KT2440overnight L-trp  75 μL (90 μg) 2386 NBRC 12996 overnight L-trp  42 μL(200 μg) 2382 NBRC 12996 overnight L-trp  21 μL (100 μg) 1706 P.taetrolens overnight L-trp 108 μL (200 μg) 2029 P. taetrolens overnightL-trp  54 μL (100 μg) 2099

The non-linearity of the overnight sample in this case is probably dueto the fact that the reactions are reaching equilibrium. Clearly, the P.taetrolens BAR has significant activity for racemization of tryptophan,as do the 12996 BAR and KT2440 BAR. It appears that the KT2440 BAR andthe P. taetrolens BAR have similar activity, which is slightly higherthan the 12996 BAR.

The DNA Sequence of the P. taetrolens arginine racemase is shown belowas SEQ ID NO:410. The PCR sequence gave two changes as compared with thepublished NCBI sequence. Specifically, the PCR sequence contained anadenosine rather than a guanine at position 902 and a cytosine ratherthan a guanine at position 921. These DNA changes resulted in one silentmutation as well as one change from glycine to aspartate at amino acidposition 301.

(SEQ ID NO: 410) ATGCCCTTCTCCCGTACCCTGCTCGCCCTTTCCCTTGGCATGGCATTGCTGCAAAACCCGGCCTTTGCTGCGCCACCCCTGTCGATGACCGACGGCGTAGCTCAAGTGAATACCCAGGACAGCAATGCCTGGGTCGAAATCAATAAAGCCGCGTTCGAGCACAACATACGGACTCTGCAAACCGCCCTCGCCGGCAAGTCGCAGATCTGCGCCGTACTCAAGGCGGATGCCTATGGCCACGGTATCGGCTTGTTGATGCCCTCGGTGATCGCCATGGGTGTTCCCTGTGTCGGTGTCGCCAGCAACGAAGAAGCCCGCGTCGTGCGCGAGAGCGGTTTCAAGGGTCAACTGATACGCGTGCGCACCGCTGCCCTGAGCGAACTGGAAGCTGCACTGCCGTACAACATGGAAGAGCTGGTGGGCAACCTGGACTTCGCGGTCAAGGCCAGCCTGATTGCCGAGGATCACGGTCGCCCGCTGGTGGTGCACCTGGGTCTGAATTCCAGCGGCATGAGCCGTAACGGAGTGGACATGACCACCGCTCAGGGCCGTCGTGATGCGGTAGCTATCACCAAGGTGCCAAACCTGGAAGTGCGGGCGATCATGACCCACTTCGCGGTCGAAGATGCTGCCGACGTGCGTGCCGGGCTCAAGGCCTTCAATCAGCAAGCCCAATGGCTGATGAACGTGGCCCAGCTTGATCGCAGCAAGATCACCCTGCACGCGGCCAACTCGTTCGCCACACTGGAGGTGCCCGAATCGCATCTGGACATGGTCCGCCCCGGCGGCGCGCTGTTCGGCGACACCGTACCGTCCCACACCGAGTACAAGCGGGTCATGCAGTTCAAGTCCCACGTGGCGTCGGTCAACAGCTACCCCAAGGGCAACACCGTCGGTTATGACCGCACGTACACCCTGGGCCGCGACTCGCGGCTGGCCAACATCACCGTCGGCTACTCTGACGGCTACCGCCGCGCGTTTACCAATAAAGGGATTGTGCTGATCAACGGCCATCGCGTGCCAGTGGTGGGCAAAGTCTCGATGAACACCCTGATGGTGGACGTCACTGACGCGCCGGATGTGAAAAGCGGCGATGAAGTGGTGCTGTTCGGGCACCAGGGCAAGGCCGAGATTACCCAGGCTGAGATCGAAGACATCAACGGTGCACTGCTTGCGGATCTGTATACCGTGTGGGGCAATTCCAACCCTAAAAT CCTGAAAGATCAGTAA.

The protein encoded by the gene of SEQ ID NO:410 was analyzed by thesignal peptide prediction program Signal P 3.0(www.cbs.dtu.dk/services/SignalP/) and a leader sequence of 23 aminoacids was predicted.

1384M Mutagenesis of P. taetrolens BAR

Mutagenesis was done using the QuickChange-Multi site-directedmutagenesis kit (Stratagene, La Jolla, Calif.), using the P. taetrolensBAR gene in pET30 which results in an untagged protein. The followingmutagenic primer was used to make the 1384M change:5′-TACCCAGGCTGAGATGGAAGACATCAACG-3′ (SEQ ID NO:411).

The site-directed mutagenesis was done as described in themanufacturer's protocol. Several isolates were sequenced (Agencourt,Beverly, Mass.) and an isolate with the correct sequence was selectedand used for further analysis.

The plasmid was transformed into BL21(DE3) cells (Novagen, Madison,Wis.). Recombinant protein was produced in Overnight Express II medium(Novagen, Madison, Wis.) containing 50 μg/mL kanamycin according tomanufacturer's protocols. Cell-free extracts were prepared usingBugBuster (Novagen, Madison, Wis.) according to manufacturer'sprotocols, desalted, and analyzed for percent expression of the targetprotein using the Experion method described above.

Total protein assays were done using a Pierce BCA kit (Pierce, Rockford,Ill.). Tryptophan racemase assays with the mutant enzyme were performedusing the wild-type enzyme prepared in the same manner as a positivecontrol. Assays contained per mL: 30 mM L-tryptophan, 50 mM potassiumphosphate pH 8, 10 μM PLP, and approximately 100 μg of racemase proteinin a cell free extract. In the case where 100 μg was not used (based onExperion % expression and Pierce total protein numbers), the resultswere normalized. Zero, 30 minute, 2 hour, and overnight samples werecollected, treated with 2% formic acid, filtered, and diluted 1:10 foranalysis using the chiral amino acid method described in Example 1.

The wild-type enzyme appeared to produce 49.1 ppm D-tryptophan in 30minutes, whereas the 1384M mutant produced 108 ppm. The 2 hour timepoint was similar—229.4 ppm D-tryptophan was produced by the wild-typeenzyme versus 541.7 for the 1384M mutant. The 1384M mutation appears tohave approximately doubled the activity of the P. taetrolens BAR. Theovernight time point for the mutant is also higher, but as the reactionsapproach equilibrium the difference between activities is reduced. Whenassays were done for monatin production as in Example 28, the 1384M didnot appear to provide any benefit over the wild-type P. taetrolensenzyme.

Example 30

A. caviae Extract Assay

Aeromonas caviae ATCC 14486 was grown in nutrient broth at 37° C. Cellsfrom the culture (200 mL) were centrifuged and washed once with 0.85%NaCl. Cell pellets were resuspended in 5 mL/g wet cell weight ofBugBuster™ (Novagen, Madison, Wis.) reagent containing 5 μL/mL proteaseinhibitor cocktail set #3 (Calbiochem-Novabiochem Corp., San Diego,Calif.) and 1 μL/mL benzonase nuclease. Samples were incubated at roomtemperature for 20 minutes on an orbital shaker. Insoluble cell debriswas removed by centrifugation at 16,000×g for 20 minutes at 4° C.Cell-free extract was desalted on a PD-10 column (GE Healthcare,Piscataway, N.J.).

Cell-free extract was assayed for tryptophan racemase activity using thefollowing protocol. One mL reactions were carried out in 50 mM potassiumphosphate (pH 8.0), 0.05 mM PLP and 30 mM L tryptophan. The reactionswere initiated by the addition of cell free extract (either 100 μL or500 μL) and were incubated at 30° C. overnight. Sample aliquots weretaken at 2 hours and after overnight incubation (zero minute samplesserved as control reactions). Concentrated formic acid (5 μL) was addedto each 250 μL sample aliquot to stop the reaction and the precipitatedprotein was removed by centrifugation. Supernatants were removed andfrozen at −80° C. until they were analyzed for D-tryptophan by thechiral amino acid method described in Example 1.

The assay results from cell extracts of A. caviae demonstrated racemaseactivity on L-tryptophan, as shown in Table 47.

TABLE 47 D-trp conc Treatment Time Point substrate Racemase extract(μg/mL) A. caviae 0 L-trp 100 μL nd A. caviae 0 L-trp 500 μL nd A.caviae 2 hr L-trp 100 μL 2 A. caviae 2 hr L-trp 500 μL 19 A. caviaeovernight L-trp 100 μL 45 A. caviae overnight L-trp 500 μL 130

After finding activity in the A. caviae cell extracts, degenerateprimers were designed (based on conserved regions of known BAR homologs)to obtain the BAR gene from this species. Degenerate primer sequencesare shown below:

Aer deg F2: (SEQ ID NO: 412) 5′-GCCAGCAACGARGARGCMCGCGT-3′; and Aer degR1: (SEQ ID NO: 413) 5′-TGGCCSTKGATCAGCACA-3′wherein K indicates G or T, R indicates A or G, S indicates C or G, andM indicates A or C.

The above primers were used to PCR amplify a 715 bp DNA fragment from A.caviae (ATCC 14486) genomic DNA. The following PCR protocol was used: A50 μL reaction contained 0.5 μL template (˜100 ng of A. caviae genomicDNA), 1.6 μM of each primer, 0.3 mM each dNTP, 10 U rT^(th) PolymeraseXL (Applied Biosystems, Foster City, Calif.), 1×XL buffer, 1 mM Mg(OAc)₂and 2.5 μL dimethyl sulfoxide. The thermocycler program used included ahot start at 94° C. for 3 minutes and 30 repetitions of the followingsteps: 94° C. for 30 seconds, 53° C. for 30 seconds, and 68° C. for 2minutes. After the 30 repetitions, the sample was maintained at 68° C.for 7 minutes and then stored at 4° C. This PCR protocol produced aproduct of 715 bp.

Cloning

The PCR product was gel purified from 0.8% TAE-agarose gel using theQiagen gel extraction kit (Qiagen, Valencia, Calif.). The product wasTOPO cloned and transformed into TOP 10 cells according tomanufacturer's protocol (Invitrogen, Carlsbad, Calif.). The plasmid DNAwas purified from the resulting transformants using the Qiagen spinminiprep kit (Qiagen, Valencia, Calif.) and screened for the correctinserts by restriction digest with EcoR 1. The sequences of plasmidsappearing to have the correct insert were verified by dideoxy chaintermination DNA sequencing with universal M13 forward primers.

The DNA sequence of the A. caviae PCR product is shown below as SEQ IDNO:414), with degenerate primer sequence regions underlined:

(SEQ ID NO: 414) GCCAGCAACGARGARGCMCGCGTTGCCCGCGAGAAGGGCTTCGAAGGTCGCCTGATGCGGGTACGTGCCGCCACCCCGGATGAAGTGGAGCAGGCCCTGCCCTACAAGCTGGAGGAGCTCATCGGCAGCCTGGAGAGCGCCAAGGGGATCGCCGACATCGCCCAGCGCCATCACACCAACATCCCGGTGCACATCGGCCTGAACTCCGCCGGCATGAGCCGCAACGGCATCGATCTGCGCCAGGACGATGCCAAGGCCGATGCCCTGGCCATGCTCAAGCTCAAGGGGATCACCCCGGTCGGCATCATGACCCACTTCCCGGTGGAGGAGAAAGAGGACGTCAAGCTGGGGCTGGCCCAGTTCAAGCTGGACTACCAGTGGCTCATCGACGCCGGCAAGCTGGATCGCAGCAAGCTCACCATCCACGCCGCCAACTCCTTCGCCACCCTGGAAGTACCGGAAGCCTACTTTGACATGGTGCGCCCGGGCGGCATCATCTATGGCGACACCATTCCCTCCTACACCGAGTACAAGAAGGTGATGGCGTTCAAGACCCAGGTCGCCTCCGTCAACCACTACCCGGCGGGCAACACCGTCGGCTATGACCGCACCTTCACCCTCAAGCGCGACTCCCTGCTGGCCAACCTGCCGATGGGCTACTCCGACGGCTACCGCCGCGCCATGAGCAACA AGGCCTATGTGCTGATCMASGGCCA,wherein R indicates A or G, S indicates C or G, and M indicates A or C.

The amino acid sequence of the partial A. caviae BAR enzyme is shown asSEQ ID NO:415 below.

(SEQ ID NO: 415) ASNEEARVAREKGFEGRLMRVRAATPDEVEQALPYKLEELIGSLESAKGIADIAQRHHTNIPVHIGLNSAGMSRNGIDLRQDDAKADALAMLKLKGITPVGIMTHFPVEEKEDVKLGLAQFKLDYQWLIDAGKLDRSKLTIHAANSFATLEVPEAYFDMVRPGGIIYGDTIPSYTEYKKVMAFKTQVASVNHYPAGNTVGYDRTFTLKRDSLLANLPMGYSDGYRRAMSNKAYVLIXG wherein X is H, Q, N, or K.

The consensus protein sequence fragment of SEQ ID NO:415 above is 89%homologous at the amino acid level to the published TIGR sequence for A.hydrophila. It was expected that because the highly related Aeromonashydrophila protein exhibited broad specificity racemase activity, aswell as the A. caviae cellular extracts, the full length coding regionfor A. caviae, once obtained, would produce a racemase that also wouldhave a broad specificity with activity on tryptophan. Genome Walkermethods were utilized to obtain the full-length gene sequence of the A.caviae BAR gene shown below as SEQ ID NO:416.

(SEQ ID NO: 416) atgcacaaga aaacactgct cgcgaccctg atctttggcc tgctggccggccaggcagtc gccgccccct atctgccgct cgccgacgac caccgcaacg gtcaggaacagaccgccgcc aacgcctggc tggaagtgga tctcggcgcc ttcgagcaca acatccagaccctgaagaat cgcctcggtg acaagggccc gcagatctgc gccatcatga aggcggacgcctacggtcac ggcatcgacc tgctggtccc ttccgtggtc aaggcaggca tcccctgcatcggcatcgcc agcaacgaag aagcacgtgt tgcccgcgag aagggcttcg aaggtcgcctgatgcgggta cgtgccgcca ccccggatga agtggagcag gccctgccct acaagctggaggagctcatc ggcagcctgg agagcgccaa ggggatcgcc gacatcgccc agcgccatcacaccaacatc ccggtgcaca tcggcctgaa ctccgccggc atgagccgca acggcatcgatctgcgccag gacgatgcca aggccgatgc cctggccatg ctcaagctca aggggatcaccccggtcggc atcatgaccc acttcccggt ggaggagaaa gaggacgtca agctggggctggcccagttc aagctggact accagtggct catcgacgcc ggcaagctgg atcgcagcaagctcaccatc cacgccgcca actccttcgc caccctggaa gtaccggaag cctactttgacatggtgcgc ccgggcggca tcatctatgg cgacaccatt ccctcctaca ccgagtacaagaaggtgatg gcgttcaaga cccaggtcgc ctccgtcaac cactacccgg cgggcaacaccgtcggctat gaccgcacct tcaccctcaa gcgcgactcc ctgctggcca acctgccgatgggctactcc gacggctacc gccgcgccat gagcaacaag gcctatgtgc tgatccatggccagaaggcc cccgtcgtgg gcaagacttc catgaacacc accatggtgg acgtcaccgacatcaagggg atcaaacccg gtgacgaggt ggtcctgttc ggacgccagg gtgatgccgaggtgaaacaa tctgatctgg aggagtacaa cggtgccctc ttggcggaca tgtacaccgtctggggctat accaacccca agaagatcaa gcgctaa.

The corresponding amino acid sequence for the A. caviae native BAR isshown below as SEQ ID NO:417:

(SEQ ID NO: 417) 1 mhkktllatl ifgllagqav aapylpladd hrngqeqtaa 41nawlevdlga fehniqtlkn rlgdkgpqic aimkadaygh 81 gidllvpsvv kagipcigiasneearvare kgfegrlmrv 121 raatpdeveq alpykleeli gslesakgia diaqrhhtni161 pvhiglnsag msrngidlrq ddakadalam lklkgitpvg 201 imthfpveekedvklglaqf kldyqwlida gkldrsklti 241 haansfatle vpeayfdmvr pggiiygdtipsyteykkvm 281 afktqvasvn hypagntvgy drtftlkrds llanlpmgys 321dgyrramsnk ayvlihgqka pvvgktsmnt tmvdvtdikg 361 ikpgdevvlf grqgdaevkqsdleeyngal ladmytvwgy 401 tnpkkikr.

The first 21 N-terminal amino acid residues of SEQ ID NO:417 arepredicted to be a signal peptide using the program Signal P 3.0(www.cbs.dtu.dk/services/SignalP/). Experimental evidence confirmed thatthe expression product was secreted into the periplasm of E. coli, andthe signal peptide was cleaved as predicted. The full length gene, whencloned and expressed using methods described above, was found to haveactivity comparable to, but greater than, the P. taetrolens BAR.

Example 31 Production of the Aldolase of SEQ ID NO: 276 in anAlternative Expression Host

The gene of SEQ ID NO: 275 was subcloned using standard molecularbiology procedures into a derivative of the pET23d vector (Novagen,Madison, Wis.) containing the E. coli metE gene and promoter inserted atthe NgoMIV restriction site and a second psil restriction site that wasadded for facile removal of the beta lactamase gene (bla). Theconstruction of this vector containing an insert for a myo-inositoloxygenase gene is described in PCT WO2006066072 in Examples 2 and 20.The aldolase insert was confirmed by DNA sequencing (AgencourtBioscience Corporation; Beverly, Mass.) and the plasmid with the correctinsert sequence was transformed into the E. coli expression hostBW30384(DE3)ΔompTΔmetE. The construction of this expression host and thetransformation protocol are also described in PCT WO2006066072 (Examples21 and 22). The aldolase gene was expressed using the Novagen OvernightExpress™ Autoinduction System II (Novagen, Madison, Wis.) containing 100mg/L ampicillin. This system was described in Example 24 for theexpression of Bacillus sphaericus (ATCC strain 10208) D-alanineaminotransferase. Cell free extracts containing the aldolase wereproduced using Novagen BugBuster™ Extraction Reagent (primary aminefree) (Novagen, Madison, Wis.) containing 1 μL/mL benzonase nuclease,0.033 μL/mL r-Lysozyme, and 5 μL/mL Protease Inhibitor Cocktail Set IIfollowing the manufacturer's recommended protocol for cell lysis. Thesoluble proteins in the cell free extracts were separated on a Bio-RadLaboratories Experion™ Automated Electrophoresis Station (Bio-Rad,Hercules, Calif.) and analyzed for percent soluble protein expressionusing the Experion Software version 1.1.98.0 as described in Example 12.

To attempt to improve the aldolase expression in E. coli, codons twothrough seven of the DNA coding sequence were mutated to changessuggested from the analysis of the wild type sequence using the RocheProteoExpert RTS E. coli HY algorithm. The changes were made using aQuikChange® Multi Site-Directed Mutagenesis Kit or QuikChange® II XLSite-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.) followingthe manufacturer's suggested protocol with 0.75 μL of Quik Solution per25 μL reaction mixture and transformation into XL10-Gold® ultracompetentcells. The mutations were generated using primers (each 45 nucleotidesin length) designed with Stratagene's web-based QuikChange® PrimerDesign Program available on-line at www. stratagene.com.qcprimerdesign.

TABLE 48 Codon 1 Codon 2 Codon 3 Codon 4 Codon 5 Codon 6 Codon 7 Wildtype ATG CCT ATC GTT GTT ACG AAG ProteoExpert #1 ATG CCA ATT GTT GTA ACTAAA ProteoExpert #2 ATG CCA ATT GTT GTT ACT AAA ProteoExpert #6 ATG CCAATT GTT GTA ACC AAA ProteoExpert #10 ATG CCA ATT GTT GTT ACC AAA

The aldolase gene sequences with the above codon changes weretransformed into the E. coli expression host BW30384(DE3)ΔompTΔmetE andthen expressed using Novagen Overnight Express™ Autoinduction System II(Novagen, Madison, Wis.). None of these mutations resulted in higherlevels of gene expression when compared to the wild type sequence.Typical results from cell free extracts analyzed by the Bio-Rad ExperionPro260 software 1.1.98.0 are shown in Table 49 below, in which thecolumn entitled Protein Expression shows values for % total solubleprotein.

The cell free extracts (0.025 mg/mL soluble protein per assay) wereassayed for their ability to produce R,R-monatin from 200 mM sodiumpyruvate and 100 mM D-tryptophan using the protocol described in Example7. The reactions (7 mL total volume) were carried out in 14 mLpolypropylene tubes in an anaerobic glove box using purified B.sphaericus (ATCC 10208) D-aminotransferase at 2 mg/mL finalconcentration. The concentrations of monatin produced at 1, 4 and 20 hafter the addition of the enzymes are shown in Table 49 below. Table 49shows that the cell free extract generated from the construct containingthe wild type aldolase sequence produced a slightly higher concentrationof monatin at 20 hours than the cell free extracts from the constructscarrying the ProteoExpert mutations. The monatin concentrations weredetermined by the LC/MS/MS method described in Example 1.

TABLE 49 Protein [Monatin] [Monatin]; [Monatin] Expression (%) mM-1 hrmM-4 hr mM-20 hr Wild type 22 2.1 4.2 13.4 ProteoExpert #1 18 1.3 3.112.4 ProteoExpert #2 18 2.1 4.7 11.6 ProteoExpert #6 18 1.4 1.9 12.4ProteoExpert #10 20 1.8 1.8 11.7

These data demonstrate that the aldolase of SEQ ID NO: 276 can beproduced in an alternative expression host without IPTG induction.

The bla gene was removed from the vector and subsequent fermentations toproduce aldolase were done without the use of antibiotic as described inPCT WO2006066072 for another enzyme. Expression levels in fed-batchfermentations at 30° C. reached a maximum at 6-8 hours post-induction,producing the aldolase of SEQ ID NO: 276 at 25-30% of the solubleprotein, according to Experion data. Stability studies showed noapparent loss of aldolase activity when the fermentation product wasleft for 6 hours at 15 or 30° C. (under both oxygen limiting conditionsas well as aerated conditions) prior to cell concentration anddisruption. The aldolase was found to be equally stable stored as eithera cell free extract or as a cell pellet when stored for 5 days at −80°C. Washing the cell pellet in buffer prior to storage at −80° C. was notrequired and actually caused a slight decrease in activity. Cellsresuspended in distilled water, fermentation supernatant, or 100 mMpotassium phosphate buffer (pH 7.8) were found to have no loss inactivity or protein concentration (judged by SDS-PAGE) when stored for11 days at room temperature or 4° C. Cell-free extract produced inpotassium phosphate buffer showed no loss of aldolase activity whenstored at 4° C. or room temperature for 5 days. Cells can be broken openin phosphate buffer, up to 25% culture supernatant or water withcomparable recovery of aldolase activity; however, addition of 1 mMMgCl₂ to water was found to slightly improve the aldolase activity.These data show that the aldolase protein is sufficiently stable to becommercially useful.

A number of embodiments of the invention have been described. Theembodiments of the invention include one or more of the above describedaspects. It will be understood that various modifications may be madewithout departing from the spirit and scope in accordance with theinvention. Accordingly, other embodiments are within the scope of thefollowing claims.

We claim:
 1. An isolated, synthetic, or recombinant nucleic acid thatencodes a polypeptide having aldolase activity, wherein the nucleic acidis selected from the group consisting of: (a) a variant nucleic acidsequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more or complete (100%) sequenceidentity to the full length of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ IDNO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ IDNO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ IDNO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ IDNO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125,SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ IDNO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153,SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ IDNO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181,SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ IDNO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209,SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ IDNO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237,SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ IDNO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265,SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ ID NO:273, SEQ IDNO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQID NO:285, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293,SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ IDNO:303, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321,SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ IDNO:331, SEQ ID NO:333, SEQ ID NO:335, SEQ ID NO:336, SEQ ID NO:337wherein the variant nucleic acid encodes the polypeptide having analdolase activity; (b) a nucleic acid comprising the full lengthsequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117,SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ IDNO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145,SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ IDNO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173,SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ IDNO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201,SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ IDNO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229,SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ IDNO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257,SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ IDNO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:285,SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ IDNO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ ID NO:313,SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321, SEQ IDNO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQID NO:333, SEQ ID NO:335, SEQ ID NO:336, SEQ ID NO:337 and/or SEQ IDNO:338, (c) a nucleic acid encoding the polypeptide having an aldolaseactivity, wherein the polypeptide is the full length sequence of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52,SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62,SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72,SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82,SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92,SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102,SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ IDNO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130,SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ IDNO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158,SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ IDNO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186,SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ IDNO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214,SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ IDNO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242,SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ IDNO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258, SEQ ID NO:260, SEQID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQ ID NO:268, SEQ ID NO:270,SEQ ID NO:272, SEQ ID NO:274, SEQ ID NO:276, SEQ ID NO:278, SEQ IDNO:280, SEQ ID NO:282, SEQ ID NO:284, SEQ ID NO:286, SEQ ID NO:288, SEQID NO:290, SEQ ID NO:292, SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298,SEQ ID NO:300, SEQ ID NO:302, SEQ ID NO:304, SEQ ID NO:306, SEQ IDNO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326,SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, and/or SEQ ID NO:334. 2.(canceled)
 3. The nucleic acid of claim 1, wherein the aldolase activitycomprises a pyruvate aldolase activity.
 4. The nucleic acid of claim 1,wherein the aldolase activity comprises a HMG aldolase activity.
 5. Thenucleic acid of claim 1, wherein the aldolase activity comprises a KHGaldolase activity. 6-16. (canceled)
 17. An expression cassette, avector, or a cloning vehicle comprising the nucleic acid of claim 1,wherein optionally the cloning vehicle comprises a viral vector, aplasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage or anartificial chromosome.
 18. The cloning vehicle of claim 17, wherein theviral vector comprises an adenovirus vector, a retroviral vector or anadeno-associated viral vector, or, the artificial chromosome comprises abacterial artificial chromosome (BAC), a bacteriophage P1-derived vector(PAC), a yeast artificial chromosome (YAC), or a mammalian artificialchromosome (MAC).
 19. A transformed cell comprising the nucleic acid ofclaim 1 wherein optionally the cell is a bacterial cell, a mammaliancell, a fungal cell, a yeast cell, an insect cell, or a plant cell. 20.A transgenic non-human animal comprising the nucleic acid of claim 1wherein optionally the transgenic non-human animal is a mouse, a rat, arabbit, a sheep, a pig, a chicken, a goat, a fish, or a cow.
 21. Atransgenic plant, plant part, or seed comprising the nucleic acid ofclaim 1, wherein optionally the plant is a corn plant, a sorghum plant,a potato plant, a tomato plant, a wheat plant, an oilseed plant, arapeseed plant, a soybean plant, a rice plant, a barley plant, a grass,a cotton plant, a cottonseed plant, a palm, a sesame plant, a peanutplant, a sunflower plant, or a tobacco plant. 22-42. (canceled)
 43. Amethod of producing the recombinant polypeptide having aldolase activitycomprising the steps of: (a) providing the nucleic acid of claim 1operably linked to a promoter; and (b) expressing the nucleic acid ofstep (a) under conditions that allow expression of the polypeptide,thereby producing recombinant polypeptide having aldolase activity,wherein optionally the method further comprises transforming a host cellwith the nucleic acid of step (a) followed by expressing the nucleicacid of step (a), thereby producing a recombinant polypeptide in atransformed cell. 44-55. (canceled)
 56. A method of generating a variantof the nucleic acid of claim 1 comprising: (a) providing a templatenucleic acid comprising the nucleic acid of claim 1; and (b) modifying,deleting, or adding one or more nucleotides in the template sequence, ora combination thereof, to generate a variant of the template nucleicacid wherein optionally the method further comprises expressing thevariant nucleic acid to generate a variant aldolase polypeptide, andoptionally the modifications, additions or deletions are introduced by amethod comprising error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, GeneSite Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR),recombination, recursive sequence recombination, phosphothioate-modifiedDNA mutagenesis, uracil-containing template mutagenesis, gapped duplexmutagenesis, point mismatch repair mutagenesis, repair-deficient hoststrain mutagenesis, chemical mutagenesis, radiogenic mutagenesis,deletion mutagenesis, restriction-selection mutagenesis,restriction-purification mutagenesis, artificial gene synthesis,ensemble mutagenesis, chimeric nucleic acid multimer creation and acombination thereof and optionally the method is iteratively repeateduntil an aldolase having an altered or different activity or an alteredor different stability from that of a polypeptide encoded by thetemplate nucleic acid is produced.
 57. The method of claim 56, whereinthe variant aldolase polypeptide: (a) is thermotolerant, and retainssome activity after being exposed to an elevated temperature; (b) hasincreased glycosylation as compared to the aldolase-encoded by atemplate nucleic acid; or, (c) has an aldolase activity under a hightemperature, wherein the aldolase-encoded by the template nucleic acidis less active under the high temperature. 58-92. (canceled)
 93. Thenucleic acid of claim 1 encoding the polypeptide having an aldolaseactivity, wherein the polypeptide is lacking a signal sequence.
 94. Thenucleic acid of claim 1, encoding a polypeptide having an aldolaseactivity and further comprising a heterologous sequence.
 95. The nucleicacid of claim 94, wherein the heterologous sequence comprises aheterologous signal sequence.